AN IN VITRO STUDY OF INTERACTIONS BETWEEN DOXAZOSIN AND ENALAPRILAT AT VASCULAR α1-ADRENOCEPTORS

1. Isolated perfused male Sprague-Dawley rat tail artery segments were used to investigate interactions between the α-₁-adrenoceptor antagonist, doxazosin, and the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, using phenylephrine (PE) as the α₁-adrenoceptor agonist. 2. In concentrations of up to 10–⁵ mol/L, enalaprilat had no effect on arterial responses to PE. 3. Doxazosin produced a concentration-dependent competitive α₁-adrenoceptor antagonism, yielding a mean pA₂ value of 8.72. 4. In the continuous presence of 10^?6 mol/L enalaprilat, doxazosin with a pA₂ value of 9.10 was a 2.4-fold more potent α₁-adrenoceptor antagonist than in the absence of enalaprilat. 5. These results are interpreted to indicate that endogenously produced angiotensin II can modulate the activity of α₁-adrenoceptors in vascular smooth muscle.     

INVESTIGATIONS INTO THE NATURE OF α2-ADRENOCEPTORS IN RAT TAIL ARTERIES

In rat isolated perfused tail arteries, dose-response curves were established for the vasopressor effects of phenylephrine (alpha 1-adrenoceptor agonist), clonidine (alpha 1- and alpha 2-adrenoceptor agonist), clonidine in the presence of 10(-7) mol/l prazosin (alpha 2-agonist), and BHT-920 (alpha 2-agonist). The ED50 values were: phenylephrine 1.85 X 10(-10) mol; clonidine 6.3 X 10(-10) mol; clonidine + prazosin 3.2 X 10(-6) mol; BHT-920 6.1 X 10(-6) mol. The arterial reactivity to BHT-920 was stable only after 4-5 h of perfusion. Responses to BHT-920 were not antagonized by yohimbine (alpha 2-adrenoceptor antagonist) but were antagonized by low concentrations of prazosin (alpha 1-adrenoceptor antagonist). These data constitute conflicting evidence regarding the existence of alpha 2-adrenoceptors in rat tail arteries. The data are consistent with the proposal that there are two recognition sites on alpha 1-adrenoceptors; phenylephrine and BHT-920 may stimulate different sites on alpha 1-adrenoceptors.     

INVESTIGATIONS INTO INTERACTIONS BETWEEN DOXAZOSIN AND ENALAPRILAT AT α1-ADRENOCEPTORS IN ANAESTHETIZED RATS

1. In anaesthetized intact Sprague-Dawley rats, the angiotensin-converting enzyme inhibitor enalaprilat, 1 mg/kg, had no significant effect on the pressor responses to the alpha 1-adrenoceptor agonist phenylephrine (PE). Doxazosin 1 mg kg was found to be a potent alpha 1-adrenoceptor antagonist. 2. The combination of enalaprilat 1 mg/kg plus doxazosin 1 mg/kg was 3.3-fold more potent in antagonizing alpha 1-adrenoceptors than doxazosin 1 mg/kg alone. 3. After treatment of rats with deoxycorticosterone acetate (DOCA) twice weekly for 5 weeks, the alpha 1-adrenoceptor antagonist action of doxazosin in anaesthetized rats was not potentiated by enalaprilat 1 mg/kg. 4. Since DOCA treatment suppresses renin activity, these findings strongly support the hypothesis that angiotensin II can modulate the functional activity of alpha 1-adrenoceptor in vascular smooth muscle.     

FURTHER INVESTIGATIONS ON THE INTERACTION BETWEEN α-ADRENOCEPTOR ANTAGONISTS AND THE CENTRAL HYPOTENSIVE EFFECT OF CLONIDINE IN RATS, RABBITS AND DOGS

1. The interactions between five alpha-adrenoceptor blocking agents and clonidine have been studied in rats, rabbits and dogs after intracisternal injections. 2. Dibozane, ethomoxane, azapetine, dibenamine and thymoxamine reduced blood pressure in rats and an antagonism of the hypotensive effects of clonidine was detected for the first four drugs. 3. In rabbits, azapetine, dibenamine, dibozane and ethomoxane were hypotensive while thymoxamine had no effect on blood pressure. Dibozane and dibenamine reduced the hypotensive effect of clonidine. 4. In dogs, azapetine, dibozane and ethomoxane reduced blood pressure, while dibenamine induced an increase in blood pressure and thymoxamine was without effect. Only dibenamine antagonized the blood pressure lowering effect of clonidine. A definite conclusion could not be drawn with azapetine due to its long duration of action of action in both rabbits and dogs. 5. These results suggest that the central receptors involved in the hypotensive effect of clonidine differ from many other central receptors and vary according to the animal species. In addition the alpha-adrenoceptor blocking agents appear not to have a unique site and mechanism of action on central cardiovascular control.     

THE EFFECTS OF CALCIUM ANTAGONISTS ON BLOOD PRESSURE AND RESPONSES TO α-ADRENOCEPTOR AGONISTS IN HYPERTENSIVE RABBITS

The effects of the calcium antagonists verapamil and nifedipine on mean arterial blood pressure, heart rate and pressor responses to a range of alpha-adrenoceptor agonists were examined in male normotensive New Zealand white rabbits and in rabbits with perinephritis hypertension. Verapamil and nifedipine caused a greater fall in mean arterial pressure in hypertensive compared to normotensive rabbits both when the fall was expressed as an absolute and as a percentage change. Effects on heart rate were similar in normotensive and hypertensive animals. Pressor responses to phenylephrine were attenuated by nifedipine and verapamil in normotensive and hypertensive rabbits. Pressor responses to alphamethyl noradrenaline were also attenuated by nifedipine, but pressor responses to BHT 920 were not significantly altered by either calcium antagonist in normotensive or hypertensive rabbits at the dose used. Thus the calcium antagonists had a greater effect on alpha 1 - than alpha 2-adrenoceptor mediated responses in both normotensive and hypertensive rabbits. Hypertensive animals showed an increased responsiveness to phenylephrine and alphamethyl noradrenaline but not BHT 920 compared to normotensives. This difference remained after treatment with both the calcium antagonists.     

SPECTROPHOTOMETRIC STUDY OF α-ADRENOCEPTORS AFFECTING MICROCIRCULATION OF RAT SKIN

1. To determine the α-adrenergic receptor subtypes that affect the microcirculation of skin, the relative absorption (RA) spectra of the skin on the backs of rats were measured using reflectance spectrophotometric methods. We injected α-adrenergic agonists, noradrenaline (NA), phenylephrine (PE) and clonidine (CL), intravenously and determined changes in the RA value at 569 nm, one of the isosbestic points of the oxyhaemoglobin and deoxyhaemoglobin absorption. 2. NA reduced the RA value and the reduction was inhibited significantly by pretreatment with phenoxybenzamine (PBZ; P<0.01). These findings suggested that the haemoglobin content in the skin tissue decreased as a result of vasoconstriction through α-adrenoceptors. 3. NA, PE and CL produced dose-dependent reductions in RA. CL and NA produced virtually equipotent reductions except at the highest dose used. PE produced smaller effects. The potency of these drugs in terms of changes in RA did not correlate with their potency in terms of rises in systemic blood pressure (NA ≤ PE ≥ CL). 4. Yohimbine (YO) inhibited the NA-induced reduction in RA to a greater degree than bunazosin (BU). Midaglizole, a specific α₂-adrenergic antagonist, significantly and dose dependently inhibited the NA-induced reduction in RA. 5. Although BU inhibited NA-induced reduction in RA only slightly, the effect was significant (P<0.05). BU significantly inhibited PE-induced reduction (P<0.01), but did not inhibit CL-induced reduction. 6. These observations suggest that the microcirculation of the skin of the rat is affected mainly by α₂-adrenoceptor mediated vasoconstriction. However, α₁-adrenoceptor mediated vasoconstriction also has some effect.     

HAEMODYNAMIC INTERACTIONS BETWEEN α- AND β-ADRENO-CEPTOR ANTAGONISTS IN CONSCIOUS OR ANAESTHETIZED RATS

1. The alpha-adrenoceptor antagonist, phentolamine, was administered intravenously, to conscious or anaesthetized rats, before or after the beta-adrenoceptor antagonist, propanolol. 2. The bradycardia which followed the combined administration of propranolol and phentolamine exceeded that induced by propranolol alone, yet the hypotensive effect of phentolamine was attenuated by propranolol. 3. The results are compatible with the concept that a significant part of the hypotensive effect of phentolamine is mediated by stimulation of vascular beta-adrenoceptors.     

DOPAMINE RELEASES ENDOTHELIUM-DERIVED RELAXING FACTOR VIA α2-ADRENOCEPTORS IN CANINE VESSELS: COMPARISONS BETWEEN FEMORAL ARTERIES AND VEINS

1. We investigated the role of vascular smooth muscle α-adrenoceptor subtypes in the vasoconstrictor response of femoral arteries and veins to dopamine and whether the vasoconstriction is modified by endothelium-dependent relaxation mediated via the activation of α₂-adrenoceptors in ring preparations of femoral arteries and veins from mongrel dogs. 2. Dopamine contracted both arteries and veins in a dose-dependent manner and this contraction was inhibited by pre-treatment with phentolamine or prazosin. Pretreatment with yohimbine shifted the dose-response curve for dopamine to the right in femoral veins, but not in arteries. 3. Phenylephrine contracted femoral arteries and veins in a dose-dependent manner and this contraction was inhibited by pretreatment with prazosin. 4. Clonidine produced a bell-shaped dose-response curve in femoral veins and this curve was shifted upwards by pretreatment with N^G-nitro-l -arginine (l -NNA). In contrast, femoral arteries were not affected by clonidine. N^G-Nitro-l -arginine potentiated contractile responses to dopamine in both veins and arteries. This potentiation was inhibited by yohimbine or by the removal of the endothelium in both arteries and veins. 5. These results suggest that dopamine contracts femoral arteries via stimulation of α₁-adrenoceptors and contracts femoral veins via stimulation of both α₁- and α₂-adrenoceptors and that these contractions are attenuated by the vasodilator action of dopamine via α₂-adrenoceptor-mediated release of endothelium-derived relaxing factor.     

CENTRAL α-ADRENOCEPTORS AND BLOOD PRESSURE REGULATION IN THE RAT

1. Radioligand binding studies using [³H]-prazosin (α₁) and [³H]-yohimbine (α₂) were undertaken to characterize central α-adrenoceptors in regions involved in blood pressure regulation. 2. Both α₁- and α₂-adrenoceptors were identified in the anterior and posterior hypothalamus and dorsal midline area of the caudal medulla oblongata of the rat. 3. The α₁- or α₂-adrenergic pharmacological specificity of the adrenoceptors was similar in each region, although their concentrations were different. 4. The concentration of α₂-adrenoceptors in the anterior hypothalamus was decreased following sino-aortic denervation suggesting that some hypothalamic α₂-adrenoceptors are associated with neurones of the baroreflex arc.     

COMPARISON OF THE ATTENUATING EFFECTS OF FOUR β-ADRENOCEPTOR AGONISTS ON RAT ISOLATED UTERUS AND AORTA

1. The ability of four 8-adrenoceptor agonists to attenuate oxytocin (0.2,2 and 20 nmol/L) or KCI (20,40 and 80 mmol/L)-induced Contractions of the uterus (n= 5–8 for each agonist) and the KCI (18 mmol/L)-induced contractions of the aorta (n = 9 for each agonist) from rats, pretreated with oestradiol has been compared. 2. Isoprenaline, salbutamol, terbutaline and procaterol (0.1–10μmol/L) attenuated the contractions of the uterus and the aorta. All four agonists had similar attenuating potencies on the uterus. 3. Procaterol caused the same maximal attenuation (33%) on the aorta as the other β-adrenoceptor agonists and is thus acting as a full β₂-adrenoceptor agonist under these experimental conditions. Isoprenaline and procaterol were much more potent than salbutamol and terbutaline in attenuating the aorta responses. 4. This study showed that isoprenaline and procaterol were potent attenuants on both the uterus and aorta whereas salbutamol and terbutaline were potent uterine but only modest aorta attenuants. This preliminary study indicates that the responsiveness of uterine and vascular tissue to certain β₂-adrenoceptors differs.     

THE ACTIONS OF SOME α-ADRENORECEPTOR AGONISTS AND ANTAGONISTS IN AN ANTINOCICEPTIVE TEST IN MICE

1. It has been shown that a number of sympathomimetic drugs, administered subcutaneously, have potent antinociceptive activity in the mouse abdominal constriction test. These drugs were found to be equieffective antagonists of the nociceptive action of acetic acid and acetylcholine. Of the drugs tested, clonidine was the most potent, being almost 60 times more active than morphine, whilst noradrenaline and oxymetazoline were approximately three and five times less active respectively than clonidine. Phenylephrine was without effect after doses of 2 mg/kg. 2. The regression lines relating the magnitude of the antinociceptive effect of noradrenaline and oxymetazoline to log dose were moved to the right in an approximately parallel manner after subcutaneous administration of piperoxane, 8 and 16 mg/kg. The antinociceptive action of clonidine was also antagonised by piperoxane, but to a lesser extent. Phentolamine had no antagonistic effect on any of these three a-agonists after subcutaneous doses of 16 mg/kg. 3. Oxymetazoline and clonidine, when given by intracisternal injection, were approximately eight-three and fifty times more potent respectively than by subcutaneous administration, and their antinociceptive action was not antagonized by piperoxane, 16 mg/kg subcutaneously, or 50 μg/kg intracisternally. 4. It is postulated that the antinociceptive action of the a-agonists is due to an effect on α-adrenoreceptors located on sensory nerve endings in the peritoneum, and that the affinities of these receptors for the α-agonists and antagonists is different from that shown by either pre- or postsynaptic α-adrenoreceptors. It also appears likely that the antinociceptive action of clonidine and oxymetazoline when given by intracisternal injection involves different mechanisms from their peripheral effects.     

IMPORTANCE OF ENDOGENOUS VASOPRESSIN IN THE HYPERTENSIVE AND BRADYCARDIC RESPONSE TO CENTRAL α1-ADRENERGIC STIMULATION

To determine the interaction between alpha 1-adrenergic and vasopressinergic mechanisms in the central regulation of cardiovascular functions, the effects of intracerebroventricular (i.c.v.) administration of the alpha 1-agonists, methoxamine and phenylephrine, were examined in conscious Long-Evans (LE) rats and Brattleboro rats with hereditary hypothalamic diabetes insipidus (DI). In LE rats, i.c.v. methoxamine and phenylephrine (3-30 micrograms/kg) increased blood pressure and decreased heart rate in a dose-related manner, while they had no detectable cardiovascular effects in DI rats. Neither i.c.v. (0.5 ng/kg per min, 1 h) nor intravenous (i.v., 2 ng/kg per min, 2 h) infusion of vasopressin (AVP) restored the cardiovascular response to i.c.v. phenylephrine in DI rats. In LE rats, however, i.v. pretreatment with the specific antagonist to the pressor effect of AVP, d(CH2)5Tyr(Me)AVP (10 micrograms/kg), attenuated the hypertensive and bradycardic effects of i.c.v. phenylephrine, while i.c.v. pretreatment with AVP antagonist (300 ng/kg) did not alter the cardiovascular response to i.c.v. alpha 1-agonist. The cardiovascular response to i.c.v. phenylephrine was blocked by i.c.v. pretreatment with the alpha 1-antagonist, prazosin (2 micrograms/kg). Intracerebroventricular phenylephrine increased plasma AVP levels 14-fold without affecting plasma angiotensin II levels. The present study clearly demonstrated that endogenous AVP plays a significant role in the cardiovascular response to i.c.v. alpha 1-agonist.     

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.     

THE EFFECT OF PRAZOSIN ON PRE- AND POSTSYNAPTIC α-RECEPTORS IN THE PITHED RAT

1. Prazosin had a marked inhibitory effect on post-synaptic a-receptors in the pithed rat, as measured by the blood pressure response to the cumulative administration of clonidine. 2. Prazosin had no inhibitory effect, however, on the pre-synaptic a-receptor, as measured by the clonidine-induced reduction in the tachycardia produced by stimulation of the cardiac nerves. 3. These results suggest that prazosin has a selective antagonistic affinity for post-synaptic a-receptors.     

DIFFERENTIAL ACTIVATION OF ADRENOCEPTOR SUBTYPES BY NORADRENALINE APPLIED FROM THE INTIMAL OR ADVENTITIAL SURFACES OF RAT ISOLATED TAIL ARTERY

• 1 The vasoconstrictor effects of noradrenaline applied to the intimal and adventitial surfaces of perfused segments of rat tail artery in the presence and absence of endothelium were studied.
• 2 Noradrenaline was about six times more potent as a vasoconstrictor when applied to the intimal than to the adventitial surface. Cocaine (25 μmol/L) enhanced responses to adventitial noradrenaline to a greater extent than those to intimal noradrenaline. A high concentration of propranolol (1 μmol/L) had a similar effect.
• 3 The vasoconstriction elicited by adventitial noradrenaline declined from a peak whereas that to intimal noradrenaline remained steady. A low concentration of propranolol (0.1 μmol/L) abolished the decline in the response to adventitial noradrenaline.
• 4 The α₁- and α₂-adrenoceptor antagonists prazosin (1 nmol/L) and idazoxan (100 nmol/L) significantly reduced responses to intimal and adventitial noradrenaline in the presence or absence of endothelium.
• 5 Removal of endothelium enhanced responses to intimal but not adventitial noradrenaline. Idazoxan produced a significantly greater reduction of responses to noradrenaline in the absence than in the presence of endothelium, and was more effective against intimal than adventitial noradrenaline. Similar effects were produced by the nitric oxide synthase inhibitor l-NAME (30 μmol/L).
• 6 It was concluded that noradrenaline acts on both α₁- and α₂-adrenoceptors to produce vasoconstriction: the α₁-adrenoceptors appear to be uniformly distributed, whereas α₂-adrenoceptors are located nearer the intima. Intimal noradrenaline also acts on endothelial α₂-adrenoceptors to release EDRF which counteracts the vasoconstrictor action of noradrenaline. Adventitial noradrenaline also acts on β-adrenoceptors located near the adventitia to counteract the vasoconstriction produced by activation of α-adrenoceptors and its action is reduced by neuronal uptake.

     

TRAIN-OF-FOUR FADE DURING NEUROMUSCULAR BLOCKADE INDUCED BY TUBOCURARINE, SUCCINYLCHOLINE OR α-BUNGAROTOXIN IN THE RAT ISOLATED HEMIDIAPHRAGM

1. Nerve-evoked maximal twitches (T1, T2, T3, T4) of the rat isolated hemidiaphragm to train-of-four (TOF) stimulation (2 Hz X 2 s) were recorded continuously in the absence or presence of tubocurarine (1.5 mumol/l), succinylcholine (40 mumol/l) or alpha-bungarotoxin (1 mumol/l). The T1 and T4 response-time profiles for the three drugs were analysed with respect to amplitude depression and the TOF ratio (T4/T1) during the development of and recovery from neuromuscular blockade. 2. Tubocurarine produced T1 block accompanied by intense TOF fade; for the same degree of T1 block, the TOF ratio was lower during the recovery from blockade after washing out tubocurarine from the bath than during the onset of blockade. There was also a correspondingly slower recovery of the TOF ratio from 90% T1 block to control levels when compared with the time for complete T1 recovery. Fade and twitch tension depression were shown clearly to be separate responses, each with its own response-time profile. Fade is therefore not simply a consequence of postjunctional cholinoceptor blockade. 3. Succinylcholine produced T1 block with only moderate TOF fade; similar recovery rates from 90% T1 block to control levels were obtained for T1 and the TOF ratio. 4. alpha-Bungarotoxin produced irreversible and complete neuromuscular blockade during which TOF fade was virtually absent. 5. The results obtained in this study closely resemble those from other similar studies in animals and in humans and clearly demonstrate that the rat isolated hemidiaphragm is a suitable in vitro model for time course studies on TOF fade.     

ADRENALINE DEPLETION IN THE HYPOTHALAMUS OF THE RAT AFTER CHRONIC α-METHYLDOPA

1. Individual anterior hypothalamic-preoptic nuclei were dissected from the brains of control and α-methyldopa treated (2 × 40 mg/kg, 5 days) rats, and the concentrations of adrenaline and other catecholamines were estimated. 2. By a combination of radioenzymatic assay and paper chromatography the concentrations of adrenaline, noradrenaline, dopamine, α-methylnoradrenaline and α-methyldopamine were determined concurrently in the same sample. 3. α-Methyldopa reduced the adrenaline levels of the hypothalamic nuclei by 47–68% of control concentrations. 4. A differential displacement of the parent catecholamines was observed and the depletions were ranked: adrenaline < dopamine < noradrenaline. 5. α-Methylnoradrenaline and α-methyldopamine were found in all hypothalamic nuclei. 6. The depletion of hypothalamic adrenaline by α-methyldopa could represent a pharmacological mechanism contributing to the antihypertensive action of the drug.