POSITIVE CHRONOTROPIC RESPONSES PRODUCED BY α-ADRENORECEPTORS IN THE PITHED RAT

• 1 Phenylephrine (1–100 γg/kg, intravenously) produced dose-dependent increases in heart rate and blood pressure in the pithed rat.
• 2 The positive chronotropic response to phenylephrine (10 μg/kg) was reduced in a dose-dependent manner by propranolol (0.01–0.3 mg/kg), but higher doses of propranolol (up to 3 mg/kg) did not reduce the response by more than about 50%. The residual response was virtually abolished by phentolamine (0.3 mg/kg) or prazosin (3 μg/kg). Labetalol (3 mg/kg) which has both α- and β-blocking activity, also abolished the positive chronotropic response.
• 3 The pressor response to phenylephrine (1–30 μg/kg) was enhanced by propranolol (1 mg/kg) and abolished by phentolamine (1 mg/kg) and prazosin (30 μg/kg). Labetalol (3 mg/kg) reduced the response to phenylephrine by 73%.
• 4 Propranolol (0.3 mg/kg) completely blocked the chronotropic and vasodepressor effects of isoprenaline (0.1 μg/kg).
• 5 It is concluded that phenylephrine acts on both α₁- and β₁-adrenoreceptors to produce an increase in heart rate, on α₁-adrenoreceptors to produce vasoconstriction and on β₂-adrenoreceptors to produce vasodilation. This latter effect is usually masked by the predominant vasoconstrictor action.

     

THE CHRONIC EFFECTS OF β-ADRENORECEPTOR ANTAGONISTS ON THE EFFLUX OF TRITIATED NORADRENALINE FROM SYMPATHETIC NERVES OF THE PITHED RAT

• 1 The effect of chronic administration of two β-adrenoreceptor blocking drugs, propranolol and timolol, on transmitter noradrenaline release from sympathetic nerves has been investigated in vivo using the pithed rat preparation.
• 2 Oral treatment for 4 weeks with either propranolol (46.3 mg/kg/day) or timolol (7.1 mg/kg/day) significantly raised the stimulation frequency threshold for release of radioactivity on stimulation of the whole spinal outflow of the pithed rat after i.v. injection of ³H-NA.
• 3 No differences in the stimulation-evoked rise in mean arterial pressure were observed between control or treated rats nor was the heart rate response to stimulation altered after timolol treatment. However, in propranolol treated rats the mean rises in heart rate were significantly higher with 3 and 30 Hz stimulation than in control rats.
• 4 Timolol treatment significantly increased the blood concentration and lowered the heart content of ³H-NA whilst propranolol treatment did not significantly change either blood or heart levels.
• 5 Log dose-response curves for mean rises in heart rate after i.v. isoprenaline were not shifted to the right in either propranolol or timolol treated pithed rats. With the lowest doses of isoprenaline (1 and 25 ng), the mean rises in heart rate in timolol treated rats were significantly greater than in the controls.
• 6 Thus chronic administration of propranolol or timolol decreased the stimulation-induced increase in plasma ³H-NA but this change in release was not related to reduced rises in blood pressure or heart rate.

     

INVOLVEMENT OF α1-ADRENOCEPTORS IN CHRONOTROPIC RESPONSES TO ENDOGENOUSLY RELEASED AMINES IN THE PITHED RAT

1. In pithed rats, yohimbine (1 mg/kg i.v.) enhanced the positive chronotropic responses to spinal stimulation of cardiac sympathetic nerves with eight pulses delivered at 2 or 4 Hz, indicating that auto-inhibition was operating, but did not increase responses to shorter lengths of trains of 8 pulses at 8, 16 or 32 Hz which did not allow sufficient time for auto-inhibition to come into effect. 2. The positive chronotropic response to cardiac sympathetic nerve stimulation with eight pulses at 8 Hz of about 60 beats/min was not affected by prazosin (1 mg/kg) or diltiazem (0.2 mg/kg), but was reduced to about 20% of the control value by propranolol (1 mg/kg). 3. In the presence of propranolol, the residual positive chronotropic responses to cardiac sympathetic nerve stimulation were virtually abolished by prazosin (1 mg/kg) or diltiazem (0.2 mg/kg). 4. The positive chronotropic response to tyramine (0.1 mg/kg i.v.) was reduced from 100 to 12 beats/min by propranolol (1 mg/kg), and the residual response was abolished by prazosin. 5. The findings indicate that noradrenaline released from cardiac sympathetic terminals by nerve stimulation or by tyramine acts on alpha 1-adrenoceptors to produce a positive chronotropic response that is revealed when beta-adrenoceptors are blocked.     

PHARMACOLOGICAL CHARACTERIZATION OF α-ADRENORECEPTOR SUBTYPES IN RAT ISOLATED THORACIC AORTA

• 1 The subtype of α-adrenoreceptor mediating contraction in rat isolated thoracic aorta was classified pharmacologically using preferential agonists and antagonists, and by utilizing mixed agonist and antagonist interactions.
• 2 Noradrenaline was 8 to 10-times more potent at contracting the aorta than phenylephrine and both agonists were about 1000 and 10,000-fold respectively more potent than azepexole (a preferential α2,-agonist).
• 3 Prazosin (a preferential α₁,-antagonist) inhibited the dose-response curves to noradrenaline and phenylephrine 100 and 1000-times respectively more effectively than either phentolamine or rauwolscine (a preferential α₂-antagonist). Furthermore, prazosin (5 times 10^-9M). completely abolished contractions elicited by a single concentration of azepexole (3 times 10^-4M).
• 4 In mixed antagonist studies, rauwolscine (5 times 10^-7M) failed to shift the dose-response curves to nor-adrenaline and phenylephrine obtained in the presence of prazosin (5 times 10^-9M).
• 5 In mixed agonist experiments, azepexole (3 times 10^-4M) acted as a partial antagonist toward phenylephrine-induced contractions.
• 6 The results suggest that the α-adrenoreceptor of the rat thoracic aorta is predominantly, if not exclusively, of the α₁-subtype.

     

RELATIVE CONTRIBUTION OF DIFFERENT VASCULAR BEDS TO THE PRESSOR EFFECTS OF α-ADRENORECEPTOR AGONISTS AND VASOPRESSIN IN PITHED RATS: RADIOACTIVE MICROSPHERE DETERMINATION

• 1 The relative fractional distribution of SICr-labelled microspheres was evaluated in pithed rats during equieffective vascoconstrictor responses evoked by infusions of the α-adrenoreceptor agonists methoxamine (α₁-selective), UK- 14,304 (α₂-selective) or vasopressin. The proportion of injected radioactive microspheres trapped in each tissue during a sustained pressor response relative to saline treated controls is considered a reflection of the degree of local vascoconstriction in the tissue analysed.
• 2 All three agonists (methoxamine, UK-14,304 and vasopressin) decreased the number of micro-spheres trapped in the mesentery and tail. Only methoxamine reduced the blood flow to the kidney and spleen. UK-14,304 did not modify the number of microspheres in the sample of skeletal muscle, however, both vasopressin and methoxamine reduced the blood flow to this tissue. Vasopressin increased the counts in the lungs and particularly in the liver but decreased the number of spheres trapped in the stomach and skin. In contrast to the a-adrenoreceptor agonists, vasopressin did not increase the number of microspheres trapped in the heart.
• 3 Since a reduction in the number of microspheres trapped in the tissue reflects a decrease in blood flow, to that organ it is reasonable to conclude that al-adrenoreceptor stimulation increases kidney, spleen, mesentery, caudal and skeletal muscle vascular resistance, whereas a2-adrenoreceptors appear to preferentially vasoconstrict the mesenteric and the caudal vascular beds.

     

FUNCTIONAL ROLE OF CARDIAC PRESYNAPTIC α2-ADRENORECEPTORS IN THE BRADYCARDIA OF α-ADRENORECEPTOR AGONISTS IN PENTOBARBITONE- AND URETHANE-ANAESTHETIZED NORMOTENSIVE RATS

• 1 The relative potencies of 6 α-adrenoreceptor agonists, with differing physicochemical properties, at cardiac presynaptic α-adrenoreceptors were determined by measuring their ability to reduce the tachycardia produced by stimulation of the cardiac sympathetic nerves in pithed rats. The compounds studied were clonidine, B-HT 933 (azepexole), oxymetazoline, St 91, naphazoline and DPI.
• 2 The bradycardia produced by the same compounds in bilaterally vagotomized, urethane- or pento-barbitone-anaesthetized normotensive rats were also compared. The relative order of presynaptic potency appeared similar to that observed for the bradycardic activity of the compounds in urethane- and pento-barbitone-anaesthetized rats. In pentobarbitone-anaesthetized rats all compounds evoked a maximal effect of 18–20% reduction in heart rate. In urethane-anaesthetized rats, however, a difference was observed between clonidine and azepexole on the one hand and oxymetazoline, St 91, naphazoline and DPI on the other hand. The former induced a 20–22% reduction in cardiac frequency, whereas the latter diminished heart rate by 10–16% only. In vagotomized, bilaterally adrenalectomized, urethane-anaesthetized rats, clonidine, St 91, naphazoline and azepexole evoked a 25% reduction in heart rate, whereas a 20% reduction was observed for DPI and oxymetazoline.
• 3 A radio-enzymatic determination of plasma catecholamines demonstrated that under urethane-anaesthesia plasma adrenaline concentrations were significantly elevated over the values observed in pentobarbitone-anaesthetized rats. This rise in plasma adrenaline was related to the amount of urethane used. In urethane-anaesthetized, bilaterally adrenalectomized rats, plasma adrenaline was not significantly elevated.
• 4 These findings demonstrate the involvement of cardiac presynaptic α₂-adrenoreceptors in the acute bradycardia, evoked by the α-adrenoreceptor agonist upon intravenous application to pentobarbitone-anaesthetized, normotensive rats. In urethane-anaesthetized rats, however, the functional role of the cardiac presynaptic α₂-adrenoreceptors may be obscured as a result of the high plasma adrenaline levels observed in these animals.

     

A STUDY ON THE POSTJUNCTIONAL EXCITATORY α-ADRENORECEPTOR SUBTYPES IN THE MESENTERIC ARTERIAL BED OF THE RAT

• 1 The nature of the postsynaptic adrenoreceptor subtypes which mediate vasoconstriction in the mesenteric arterial bed of the rat was investigated using mixed and selective α₁, α₂ and β-agonists and antagonists.
• 2 Phenylephrine (PE) an α₁selective agonist and noradrenaline (NA) a mixed α₁and α₂-agonist, produced a rise in perfusion pressure (vasoconstriction). The responses to NA remained stable with time whereas responses to PE considerably increased.
• 3 UK14304 an α₂-selective agonist at low doses (10^?8 ?10^?7 moles), caused small, slow contractions in most preparations. Repeated administration of these doses or slightly higher ones, desensitized the tissue to this compound but not to NA or PE. Finally, UK14304 given simultaneously with NA or PE, at doses higher than 5 × 10^?7 moles, reduced contractions to the latter compounds and this effect was not altered by 10^?7 M rauwolscine, an α₂-selective antagonist.
• 4 Prazosin, an α₁-selective antagonist, as expected, reduced contractions to NA considerably at 10^?10 ?10^?8 M and abolished contractions to UK14304 at 2 × 10^?9 M.
• 5 Rauwolscine, at 10^?8 M, potentiated contractions to NA and at 10^?6 M reduced contractions to both NA and PE (when compared to time controls).
• 6 When propranolol (10^?6 M), a β-antagonist was included in the perfusion fluid, rauwolscine no longer potentiated responses to NA but reduced them at all concentrations. Under the same conditions rauwolscine affected the responses to PE in a similar direction to that observed in the absence of propranolol.
• 7 These results suggest that in the rat mesenteric arterial bed:

• a. rauwolscine exerts an effect additional to α₂-adrenoreceptor antagonism. Modification of this effect by propranolol indicates an interaction between this effect of rauwolscine and the β-adrenoreceptor.
• b. vasoconstriction in the mesenteric arterial bed of the rat is mainly mediated postsynaptically by α₁-adrenoreceptors although the contribution of an α₂-mediated component cannot be excluded.
• c. UK14304 is an α₁-partial agonist as well as an α₂-agonist.

     

PHARMACOLOGICAL APPROACHES TO THE CHARACTERIZATION OF β-ADRENORECEPTOR POPULATIONS IN TISSUES

• 1 The locations of the Schild plots for a selective β-adrenoreceptor antagonist using different agonists have been used as a means of determining whether the β-adrenoreceptor population of a tissue is heterogeneous (β₁ plus β₂) or homogeneous (β₁ or β₂).
• 2 On guinea-pig trachea, Schild plots for ICI 118,551 (β₂-selective adrenoreceptor antagonist), obtained using fenoterol (β₂-selective) or noradrenaline (β₁-selective) as agonist, were separated, indicating a heterogeneous β-adrenoreceptor population. In contrast, Schild plots for a non-selective antagonist, pindolol, using the same two agonists, were superimposed.
• 3 On guinea-pig trachea, the Schild plot for ICI 118,551 with salbutamol (β₂-selective agonist) was superimposed on that with fenoterol (slopes of both plots were close to unity). The Schild plots for ICI 118,551 with adrenaline or rimiterol (β₂-selective agonists) gave pA₂ values similar to those obtained with fenoterol or salbutamol as agonist, although the slopes of these plots were less than unity. The Schild plot obtained using any of these β₂-selective agonists was clearly separated from that using noradrenaline. This was in contrast to guinea-pig atria where Schild plots were superimposed whatever the β-adrenoreceptor agonist used.
• 4 These data confirmed that, in a tissue with a heterogeneous β-adrenoreceptor population, separated Schild plots for an antagonist are obtained only if the antagonist is selective and if agonists of differing selectivity are used. This is the theoretically predictable result.
• 5 Data were also obtained (for the positive chronotropic response of guinea-pig, rat and cat atria and for the relaxation response of guinea-pig trachea) using another method for determining whether or not the β-adrenoreceptor population of a tissue is homogeneous. This method did not require the use of selective agonists.
• 6 The concentration ratios obtained for the antagonism of isoprenaline by atenolol (β₁-selective) and ICI 118,551 alone and then in combination, indicated the involvement of a single β-adrenoreceptor population in responses of guinea-pig and rat atria but not of cat atria and guinea-pig trachea. These results were consistent with conclusions from Schild plot experiments that positive chronotropic responses in rat and guinea-pig atria involve only β₁-adrenoreceptors whereas chronotropic responses in cat atria and relaxation of guinea-pig trachea involve both β₁ and β₂-adrenoreceptors.

     

COMPARISON OF THE CARDIAC EFFECTS OF β-ADRENORECEPTOR AGONISTS IN ANAESTHETISED AND CONSCIOUS DOGS

• 1 The cardiovascular effects of some β-adrenoreceptor agonists on heart rate, blood pressure and myocardial contractility (maximum rate of change of left ventricular pressure/integrated isometric tension) were measured in pentobarbitone-anaesthetised and conscious, instrumented greyhounds.
• 2 In anaesthetised dogs isoprenaline increased heart rate and myocardial contractility and reduced blood pressure. Prenalterol and RO 363, in equiactive inotropic doses, induced greater increases in heart rate than isoprenaline if blood pressure fell by less than 25 mmHg. Salbutamol had hypotensive activity at all doses and appeared to be a relatively selective inotrope.
• 3 None of the agonists caused blood pressure to fall in the conscious dogs. Prenalterol and RO 363 were more effective inotropic stimulants, producing smaller increases in heart rate and more pronounced increases in myocardial contractility. Salbutamol, however, elicited greater increases in heart rate in the conscious animals and the inotropic selectivity demonstrated in the anaesthetised animals was lost.
• 4 The direct effects of the β-adrenoreceptor agonists, without modification by reflexes could be observed in the anaesthetised animals. The differences in the actions of the agonists in the conscious animals appear to be attributable to the state of the baroreceptor reflex control system and the relatively enhanced responsiveness of the heart.

     

RESISTANCE OF SYMPATHETIC CARDIAC NERVE FUNCTION IN THE PITHED RAT TO PREJUNCTIONAL EFFECTS OF ANGIOTENSIN II

1. The spinal sympathetic outflow (C7-T2) of pithed male Wistar rats was electrically stimulated (30-50 V, 0.5 ms, 0.1-1.0 Hz) and heart rate and arterial blood pressure were recorded. D-Tubocurarine (1 mg/kg) and atropine (1 mg/kg) were administered intravenously (i.v.) to reduce voluntary and parasympathetic nerve activity. 2. Angiotensin II (0.39-3.4 micrograms/kg per min) was infused at a rate which caused a sustained rise in diastolic blood pressure of at least 25 mmHg but which did not alter basal heart rate. 3. Chronotropic responses to sympathetic nerve stimulation were not affected by infusion of angiotensin II, and were also unaltered by pretreatment of rats with indomethacin (5 mg/kg, i.v.) 10 min prior to commencement of stimulation. 4. These results suggest that positive chronotropic responses to cardiac sympathetic nerve stimulation in pithed rats are not affected by increased angiotensin II levels. Indomethacin had no effect, which indicates that cyclo-oxygenase products were not involved in modulation of chronotropic responses to cardiac sympathetic nerve stimulation.     

STUDIES ON THE PRE- AND POSTJUNCTIONAL ACTIVITIES OF α-ADRENORECEPTOR AGONISTS AND THEIR CARDIOVASCULAR EFFECTS IN THE ANAESTHETIZED RAT

• 1 The selectivity of a number of agonists for peripheral α₁- and α₂- adrenoreceptors was determined and related to their cardiovascular effects in pentobarbitone-anaesthetized rats.
• 2 In isolated tissues, presynaptic α₂- and postsynaptic α₁-adrenoreceptor activity was determined on the rat vas deferens and anococcygeus muscle respectively. Xylazine, guanabenz, guanoxabenz and guanfacin were more selective, whilst lofexidine, tiamenidine, naphazoline, oxymetazoline and St 91 were less selective than clonidine for presynaptic α₂-adrenoreceptors.
• 3 The anococcygeus muscle of the pithed rat was used to determine the α-adrenoreceptor selectivity of the compounds in vivo. The selectivities were similar to those obtained in vitro.
• 4 Following intravenous administration to pentobarbitone-anaesthetized rats guanabenz and guanfacin caused small transient pressor responses and a prolonged secondary hypotension. In contrast oxymetazoline and St 91 produced marked initial pressor responses and the secondary reduction in blood pressure was absent. Clonidine and tiamenidine produced marked initial pressor responses followed by secondary hypotensive effects. All compounds reduced heart rate.
• 5 Intracerebroventricular (i.c.v.) administration of clonidine, guanabenz and guanfacin elicited falls in blood pressure and heart rate. In contrast blood pressure was either unaffected or elevated following i.c.v. administration of St 91, oxymetazoline and tiamenidine.
• 6 It is concluded that in the normotensive rat, central α₂-adrenoreceptors play a major role in the hypotensive effects of clonidine and related compounds.

     

THE CHRONIC EFFECTS OF β-ADRENORECEPTOR BLOCKING AGENTS ON TRANSMITTER OVERFLOW IN THE ANOCOCCYGEUS MUSCLE OF THE RAT

1 The effect of chronic administration of three β-adrenoreceptor blocking agents, propranolol, timolol and atenolol on transmitter release from sympathetic nerves has been investigated in the isolated anococcygeus muscle of the rat. 2 Oral treatment for 11 weeks with either propranolol (12 mg/kg/day), or timolol (1.2 mg/kg/day) but not atenolol (12 mg/kg/day) significantly increased the stimulation-induced overflow of radioactivity following preincubation of the tissue with ³H-NA. 3 No increase in end-organ sensitivity, as measured in terms of responsiveness to exogenous acetylcholine, tyramine or NA, was observed. 4 The ineffectiveness of atenolol compared to the other β-adrenoreceptor blocking drugs may be related to the finding that, in acute experiments, atenolol inhibited tissue uptake of ³H-NA. 5 It is postulated that increased release of transmitter may be related to prolonged blockade of presynaptic β-receptors and a resultant increase in presynaptic receptor sensitivity.     

HETEROGENEITY OF β-ADRENORECEPTOR SUBTYPES IN THE HUMAN PLACENTA

• 1 The human placenta is a rich source of β-adrenoreceptors. However, previous work has provided conflicting evidence as to the predominant receptor subtype present. This study was designed to clarify this situation.
• 2 The radioligand ³H-dihydroalprenolol (³H-DHA) was used to identify β-adrenoreceptor binding sites in membranes prepared from human placentae obtained immediately post partum from normal full term pregnancies.
• 3 ³H-DHA binding to human placental membranes was saturable, of high affinity and exhibited the pharmacological characteristics of a β-adrenoreceptor. Displacement by β-adrenoreceptor agonists suggested a predominant β₁-subtype with a hierarchy of potency isoprenaline > adrenaline > noradrenaline. Analysis of the displacement of the specific binding of ³H-DHA by β-adrenoreceptor antagonists yielded Hill plots with an apparent slope (nH) of one for the non selective antagonist (?)propranolol, and of less than one for the highly selective antagonists practolol (β₁), and ICI 118,551 (β₂) indicating a possible heterogeneity of binding sites.
• 4 A direct comparison of the displacement of ³H-DHA binding by selective antagonists in the placenta with that occurring in rat and rabbit lung, further established that a mixture of β₁- and β₂-adrenoreceptor binding sites was present.
• 5 Graphical analysis by Hofstee plot and computer assisted iterative curve fitting was used to further evaluate the displacement by the selective agents and established the presence of an heterogeneous population of β-adrenoreceptor subtypes in the human placenta with approximately 65%β₁ and 35%β₂.

     

INFLUENCE OF URAPIDIL ON α- AND β-ADRENORECEPTORS IN PITHED RATS

• 1 The interaction of urapidil with pre- and postsynaptic a-adrenoreceptors and with postsynaptic 8- adrenoreceptors was studied in pithed normotensive rats and compared to the effects of clonidine, prazosin, and atenolol.
• 2 I.v. injection of urapidil did not substantially change blood pressure, while clonidine raised blood pressure.
• 3 Urapidil dose-dependently antagonized the pressor effects of the al-agonist L-phenylephrine (pDR2 6.8) and of the a2-agonist azepexole (pDR2 5.2). Compared to urapidil, prazosin was a more potent antagonist of phenylephrine at postsynaptic vascular al-adrenoreceptors (pDR2 8.7) and of azepexole at a2-adrenoreceptors (pDR2 5.6).
• 4 Urapidil inhibited the tachycardia produced by discontinuous or continuous electrical stimulation of the thoracic spinal outflows (IDSo 4.8 and 27.2 Fmol/kg, respectively). In contrast to the inhibitory action of clonidine (IDSo 0.039 and 0.023 pmolikg, respectively), the inhibition by urapidil was not reversed by the selective a2-antagonist rauwolscine (10 pmolikg). Prazosin did not change stimulation-evoked tachycardia but atenolol caused pronounced inhibition (IDS0 0.158 prnol/kg, discontinous stimulation).
• 5 Umpidi! dcse-APnendently –r antagerincd the tachycardic effect of isoprenaIinP nt g:-adrennreceptors (pDR2 5.1) but also exhibited intrinsic activity by increasing basal heart rate (maximum effect of urapidil was 30% of that of isoprenaline). Urapidil did not change the vasodilatory P2-adrenoreceptor-mediated effect of isoprenaline.
• 6 The results suggest that urapidil is an antagonist at postsynaptic vascular el- and a2-adrenoreceptors, with a greater potency against al-adrenoreceptors. An agonistic interaction of urapidil with presynaptic a2-adrenoreceptors could not be demonstrated in pithed rats. Instead, the inhibition by urapidil of stimulation-evoked tachycardia could be accounted for by its bl-adrenoreceptor antagonistic effect.