Synergistic interactions between two alpha(2)-adrenoceptor agonists, dexmedetomidine and ST-91, in two substrains of Sprague-Dawley rats

Several lines of evidence indicate that the antinociception produced by intrathecal administration of the alpha(2)-adrenoceptor agonists dexmedetomidine or ST-91 is mediated by different subtypes of the alpha(2)-adrenoceptor. We recently provided additional pharmacologic evidence for this idea, as well as for differences in the function of these receptors between Harlan and Sasco rats, two widely-used outbred substrains of Sprague-Dawley rat. The present study used isobolographic analysis to further characterize the receptors at which intrathecally administered ST-91 and dexmedetomidine act in these two substrains. The rationale for these studies derives from the assumption that if dexmedetomidine and ST-91 act as agonists at the same receptor then they should interact in an additive manner. However, if they interact in a supra-additive manner, then they must act at different subtypes of the alpha(2)-adrenoceptor. In the tail-flick test, the dose-effect relationship for a 1:3 mixture of dexmedetomidine and ST-91 was shifted significantly to the left of the theoretical dose-additive line in both Harlan and Sasco Sprague-Dawley rats. A similar finding was made in the hot-plate test despite the fact that the dose-response characteristics of the agonists were different in this test. Thus, in Harlan rats, in which ST-91 is a full agonist and dexmedetomidine is essentially inactive, the dose-effect relationship for the mixture of dexmedetomidine and ST-91 was shifted far to the left of the dose-additive line. Similarly, in Sasco rats, in which ST-91 is a partial agonist and dexmedetomidine is inactive, co-administration of the two agonists also shifted the dose-response relationship to the left of the dose-additive line. The consistent finding that these two alpha(2)-adrenoceptor agonists interact in a supra-additive manner provides strong evidence that dexmedetomidine and ST-91 produce antinociception by acting at different alpha(2)-adrenoceptor subtypes in the spinal cord. This conclusion is consistent with the earlier proposal that dexmedetomidine acts predominantly at alpha(2A)-adrenoceptors whereas ST-91 acts predominantly at non-alpha(2A)-adrenoceptors. Recent anatomical evidence indicates that these non-alpha(2A) adrenoceptors may be of the alpha(2C) type. The synergistic combination of an alpha(2A)- and an alpha(2C)-adrenoceptor agonist may provide a unique and highly effective drug combination for the treatment of pain without the sedation produced by an equianalgesic dose of a single alpha(2)-adrenoceptor agonist.     

Spinal administration of alpha 2-adrenoceptor agonists and opioids or local anaesthetic agents

The effects of combined spinal administration of alpha(2)-adrenoceptor agonists, local anaesthetics, and opioids have been extensively studied. The motor and the sensory block of spinal and epidural anaesthesia is enhanced and prolonged by the combination of clonidine with the local anaesthetics lidocaine, tetracaine and bupivacaine. Because higher plasma levels of local anaesthetics were measured when clonidine was injected epidurally, the enhancement of the local anaesthetic's effect by clonidine is not due to slowed resorption, but rather to direct spinal and supraspinal effects of clonidine. Furthermore, direct local anaesthetic properties of clonidine on nerve fibres are discussed. In addition, in children the combination of clonidine and bupivacaine for caudal anaesthesia resulted in a marked prolongation of postoperative pain relief. Circulatory effects of combined clonidine and local anaesthetics are the result of the specific spinal blockade and the central and peripheral effects of clonidine. The combined epidural and intrathecal administration of opioids and alpha(2)-adrenoceptor agonists provides better postoperative pain relief than the administration of either substance alone. In humans, the interaction seems to be additive rather than supra-additive. Because of its limited duration of action, continuous administration of clonidine is recommended, especially when it is used in combination with opioids. Neither the incidence nor the severity of side effects is increased by a combined therapy with opioids. Despite the sedative properties of clonidine, there is no increased risk of respiratory depression when clonidine is given in combination with opioids. The inhibiting effect on the sympathetic nervous system activity regularly observed during spinal administration of clonidine supports the value of this therapy and will support its use in the future. Therefore, the combination of alpha(2)-adrenoceptor agonists with local anaesthetics or opioids is reasonable and may improve anaesthetic practice.     

Beta-adrenergic agonists increase lung liquid clearance in anesthetized sheep.

We did experiments to determine whether beta-adrenergic agonists increase lung liquid clearance in anesthetized ventilated adult sheep and, if so, whether the increase is mediated by beta receptors and what mechanism is involved. We instilled 100 ml of autologous serum either alone or with a beta-adrenergic agonist (terbutaline, 10(-5) M, or epinephrine, 5.5 X 10(-6) M) into one lower lobe. After 4 h both terbutaline and epinephrine increased lung liquid clearance. The increase in lung liquid clearance was inhibited when propranolol (a beta blocker) or amiloride (a sodium channel blocker) was added to the terbutaline. Increased clearance was not explained by changes in pulmonary hemodynamics, pulmonary blood flow, or lung lymph flow. We conclude that beta-adrenergic agonists increase lung liquid clearance in anesthetized intact adult sheep. This increase is mediated through beta receptors and probably depends on increased active transport of sodium across the alveolar barrier.     

Experimental data on the interactions of alpha 2-adrenoceptor agonists with anaesthetics,analgesics and local anaesthetics

The interactions of alpha(2)-adrenoceptor agonists with anaesthetics, analgesics and local anaesthetics are not only of practical importance in many areas of anaesthesia and pain therapy, but also of significant theoretical interest. Laboratory investigations and animal research allow us to study the pharmacological mechanisms of these drug interactions by using the tools of molecular biology. This may lead to the establishment of a scientific basis allowing the definition of rational drug therapies. In the first part of this overview, recent data on local anaesthetic effects of alpha(2)-adrenoceptor agonists on peripheral nerve conduction and their interaction with local anaesthetics are presented. Some important experimental work focusing on additive and synergistic spinal interactions of alpha(2)-adrenoceptor agonists with opioids and some other agents are then discussed. Finally, there is a brief reference to the central role of the spinal cord in mediating and controlling nociception.     

Alpha 2-adrenoceptor agonists for the treatment of chronic pain

The antinociceptive effect of alpha(2)-adrenoceptor agonists is mediated by activation of descending inhibiting noradrenergic systems, which modulates 'wide-dynamic-range' neurones. Furthermore, they inhibit the liberation of substance P and endorphines and activate serotoninergic neurones. Despite this variety of antinociceptive actions, there is still little experience with alpha(2)-adrenoceptor agonists as therapeutic agents for use in chronic pain syndromes. Studies in animals and patients have shown that the transdermal, epidural and intravenous administration of the alpha(2)-adrenoceptor agonist clonidine reduces pain intensity in neuropathic pain syndromes for periods varying from some hours up to 1 month. Patients suffering from lancinating or sharp pain respond best to this therapy. Topically applied clonidine (200-300 microg) relieves hyperalgesia in sympathetically maintained pain. Epidural administration of 300 microg clonidine dissolved in 5 ml NaCl 0.9 % has also been shown to be effective. In patients suffering from cancer pain tolerant to opioids, pain control has proved possible again with combinations of opioids and clonidine. In isolated cases clonidine has been administered epidurally at a dose of 1500 microg/day for almost 5 months without evidence for any histotoxic property of clonidine. Side effects often observed during administration of alpha(2)-adrenoceptor agonists are dry mouth, sedation, hypotension and bradycardia. Therapeutic interventions are usually not required.     

Comparison of the behavioral and neurochemical effects of the two optical enantiomers of medetomidine, a selective alpha-2-adrenoceptor agonist.

Medetomidine (MED) is a veterinary sedative whose mode of action is activation of alpha-2 adrenoceptors. Because the carbon atom which separates the two ring systems is methylated, there is a center of stereochemical asymmetry in the molecule. The resulting enantiomers, d-MED and l-MED have recently become available for study. The biological activity of MED, as is now demonstrated in rats in vivo, seems to reside almost exclusively in the d-MED form. Only at extremely high doses (e.g., 10 mg/kg) does l-MED exert any effects, interpreted as alpha-2 adrenoceptor antagonism. In contrast, doses of d-MED as low as 30 micrograms/kg cause sedation, hypothermia and induce neurochemical changes in norepinephrine and 5-hydroxytryptamine metabolism in brain characteristic of alpha-2-agonists (decreases in concentrations of biogenic amine metabolites, turnover and increases in concentration of parent amine). The most sensitive neurochemical indicator of the alpha-2-agonist action of d-MED was the concentration of unconjugated 3-methoxy-4-hydroxy-phenyethylene glycol in rat cerebral spinal fluid, doses of d-MED as low as 10 micrograms/kg caused a significant reduction in this norepinephrine metabolite. Simultaneous administration of the specific alpha-2 adrenoceptor antagonist, atipamezole (1 mg/kg), effectively inhibited the behavioral and most of the neurochemical actions of d-MED (100 micrograms/kg). It is concluded that the enantiomers of MED may be extremely useful in elucidating structure action relationships at alpha-2 adrenoceptors.     

Simultaneous alpha2B- and beta2-adrenoceptor activation sensitizes the alpha2B-adrenoceptor for agonist-induced down-regulation

We recently reported that alpha(2A)-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 microM) in BE(2)-C cells that express both alpha(2)- and beta(2)-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by beta(2)-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the alpha(2A)-versus alpha(2B)-ARs have been reported, the present study examines the effects of simultaneous activation of alpha(2B)- and beta(2)-ARs on alpha(2B)-AR number and signaling. We studied NG108 cells that naturally express alpha(2B)-ARs, and BN17 cells, NG108 cells transfected to express the human beta(2)-AR. In NG108 cells, alpha(2B)-AR desensitization and down-regulation require treatment with 20 microM EPI or NE; GRK expression was not changed. In BN17 cells expressing beta(2)-ARs, the threshold EPI concentration for alpha(2B)-AR desensitization and down-regulation was reduced to 0.3 microM; 10 microM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The beta-AR antagonist alprenolol (1 microM) and GRK3 antisense (but not sense) DNA blocked 0.3 microM EPI- and 10 microM NE-induced desensitization and down-regulation of the alpha(2B)-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of alpha(2B)- and beta(2)-ARs results in a 67-fold decrease in the threshold concentration of EPI required for alpha(2B)-AR down-regulation. This lower threshold for down-regulation is associated with alpha(2B)- and beta(2)-AR dependent up-regulation of GRK3 expression.     

Changes in vascular alpha 1- and alpha 2-adrenoceptor responsiveness by selegiline treatment

Pharmacoepidemiological studies have reported an excess of mortality with selegiline, a MAO B inhibitor used in the treatment of Parkinson's disease. The mechanism of this putative adverse effect remains unknown but an interaction with the sympathetic nervous system was suggested. The aim of the present study was to investigate the influence of selegiline (10 mg/daily, orally during one week) on vascular alpha1- and alpha2-adrenoceptor responsiveness in conscious unrestrained dogs. Selegiline significantly increased resting values of both systolic and diastolic blood pressures and noradrenaline plasma levels (HPLC) without changing heart rate. Moreover, spectral analysis of systolic blood pressure (Fast Fourier Transformation) showed that selegiline increased the relative energy of a low frequency band without modifying the total spectrum. ED 50 calculated from dose-pressor response curves with phenylephrine (after beta-blockade by propranolol), an index of alpha1-adrenoceptor response or with noradrenaline (after alpha1- and beta blockade by prazosin plus propranolol), an index of alpha2-adrenoceptor response, were significantly higher after selegiline. Selegiline failed to modify the number of platelet alpha2-adrenoceptors measured by [(3)H] RX 821002 binding. Yohimbine-induced increase in noradrenaline release was significantly more marked after selegiline. These results support the evidence that selegiline induces a vascular alpha1- and alpha2-adrenoceptor-hyposensitivity that can be explained by the increase in noradrenaline release elicited by the drug.     

Relative efficacy of spinal alpha-2 agonists, dexmedetomidine, clonidine and ST-91, determined in vivo by using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, an irreversible antagonist.

To examine the relative efficacy of spinally administered alpha-2 adrenergic agonists [dexmedetomidine, clonidine and ST-91 (2-[2,6- diethylphenylamino]-2-imidazole)] on the 52.5 degrees C hot plate in rats, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline), an irreversible alpha-2 antagonist, was administered intrathecally (in doses of 0, 8.1, 81 and 810 nmol) 24 hr before the administration of these agonists. EEDQ alone results in an acute mild hyperalgesic effect. Intrathecal dexmedetomidine, clonidine and ST-91 result in a dose-dependent increase in the hot plate response latency: ED50 (95% confidence interval) = 13 (2.1-73), 120 (52-310) and 21 (1.3-240) nmol, respectively. Pretreatment with intrathecal EEDQ (8.1-810 nmol) caused a rightward shift of dose-response curve and reduction of maximal effect of alpha-2 agonists used. For example, 24 hr after 81 nmol of EEDQ, the degree of the rightward shift and the depression of slope was ST-91 greater than clonidine greater than dexmedetomidine. Analysis of the double reciprocal plot indicated that at the ED50, intrathecal dexmedetomidine, clonidine and ST-91 required an apparent occupancy of 10, 36 and 30%, respectively. These results suggest that dexmedetomidine has a higher intrinsic efficacy than clonidine or ST-91.     

A study of alpha1- and alpha2-adrenoceptor responsiveness in diabetic insipidus dogs

Summary— The present study was performed to investigate the participation of circulating vasopressin in alpha-adrenoceptor responsiveness. Thus, we compared the pressor responses induced by selective alpha1-or alpha2-adrenoceptor stimulation in two groups of conscious dogs: a) normal animals and b) animals with surgically-induced diabetes insipidus. In addition, platelet alpha2-adrenoceptors labelled with (³H)RX821002 were compared in the two groups. The pressor response to alpha1-adrenoceptor stimulation [ ie successive doses of noradrenaline (0.5, 1, 2, 4 μg/kg iv) after propranolol (1 mg/kg iv) plus yohimbine (0.5 mg/kg iv)] was significantly ( P < 0.05) less pronounced in diabetic insipidus than in normal dogs. In contrast, the magnitude of the pressor effects of alpha2-adrenoceptor stimulation [ ie noradrenaline after propranolol plus prazosin (1 mg/kg iv)] was the same in the two groups of animals. B_max and Kd values for (³H)RX821002 binding on platelets were similar in diabetic insipidus and normal dogs. This study shows that alpha1- (but not alpha2-) adrenoceptor responsiveness is decreased in diabetic insipidus suggesting the involvement of vasopressin in the mechanisms of the vascular alpha1 -adrenoceptor pressor response.     

Clinical evaluation of a benzofuroquinolizine alpha 2-adrenoceptor antagonist

The pharmacodynamics of MK-912, a benzofuroquinolizine alpha-adrenoceptor antagonist, were evaluated in healthy male volunteers. Eight subjects were treated with single oral doses of 0.1, 1.0, and 2.0 mg MK-912 and with a placebo in a four-period, double-blind, balanced, crossover study. Hemodynamic effects were observed with the 2.0 mg dose of MK-912 (peak increase from baseline in systolic and diastolic blood pressure +/- SEM, 14.8/9.2 +/- 2.9/2.1 mm Hg; peak increase in heart rate, 6.3 +/- 2.1 beats/min; p less than 0.05 versus placebo). Plasma concentrations of 3-methoxy-4-hydroxyphenylglycol (MHPG, a catecholamine metabolite) were increased 29% +/- 7% and 40% +/- 10% above baseline 2 hours after administration of 1.0 and 2.0 mg MK-912, respectively (p less than 0.01 compared with placebo). A modest dose-dependent reduction (5% to 10%) in fasting plasma glucose concentration was observed 1/2 to 1 hour after administration of 1.0 and 2.0 mg MK-912 (p less than 0.05 compared with placebo), without significant change in plasma insulin values. MK-912 was well tolerated, although it did have a mild anxiogenic effect. MK-912 is a potent, orally active agent with a pharmacologic profile consistent with alpha 2-adrenoceptor antagonism.     

The effects of alpha2-adrenoceptor agents on anti-hyperalgesic effects of carbamazepine and oxcarbazepine in a rat model of inflammatory pain

In this study, the effects of yohimbine (alpha2-adrenoceptor antagonist) and clonidine (alpha2-adrenoceptor agonist) on anti-hyperalgesia induced by carbamazepine and oxcarbazepine in a rat model of inflammatory pain were investigated. Carbamazepine (10-40 mg/kg; i.p.) and oxcarbazepine (40-160 mg/kg; i.p.) caused a significant dose-dependent reduction of the paw inflammatory hyperalgesia induced by concanavalin A (Con A, intraplantarly) in a paw pressure test in rats. Yohimbine (1-3 mg/kg; i.p.) significantly depressed the anti-hyperalgesic effects of carbamazepine and oxcarbazepine, in a dose- and time-dependent manner. Both drug mixtures (carbamazepine-clonidine and oxcarbazepine-clonidine) administered in fixed-dose fractions of the ED50 (1/2, 1/4 and 1/8) caused significant and dose-dependent reduction of the hyperalgesia induced by Con A. Isobolographic analysis revealed a significant synergistic (supra-additive) anti-hyperalgesic effect of both combinations tested. These results indicate that anti-hyperalgesic effects of carbamazepine and oxcarbazepine are, at least partially, mediated by activation of adrenergic alpha2-receptors. In addition, synergistic interaction for anti-hyperalgesia between carbamazepine and clonidine, as well as oxcarbazepine and clonidine in a model of inflammatory hyperalgesia, was demonstrated.     

Mechanism of the vascular angiotensin II/alpha2-adrenoceptor interaction

alpha(2)-Adrenoceptors potentiate vascular responses to angiotensin II. The goal of this study was to test the hypothesis that the phospholipase C (PLC)/protein kinase C (PKC)/c-src/phosphatidylinositol 3-kinase (PI3K) pathway contributes to the vascular angiotensin II/alpha(2)-adrenoceptor interaction. In rats in vivo, intrarenal infusions of angiotensin II (10 ng/kg/min) increased renal vascular resistance by 5.8 +/- 0.5 units, and this response was enhanced (p < 0.05) to 9.1 +/- 1.2 units by UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; 3 microg/kg/min; alpha(2)-adrenoceptor agonist]. Intrarenal infusions of U-73122 [1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione; 3 microg/min; PLC inhibitor], GF109203X [bisindolylmaleimide I; 10 microg/min; PKC inhibitor], CGP77675 [1-(2-{4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenyl}ethyl)piperidin-4-ol; 5 microg/min; c-src inhibitor], and wortmannin (1 microg/min; PI3K inhibitor) abolished the angiotensin II/alpha(2)-adrenoceptor interaction. In isolated perfused rat kidneys, angiotensin II (0.3, 1, and 3 nM) increased perfusion pressure (by 15 +/- 8, 39 +/- 4, and 93 +/- 9 mm Hg, respectively), and UK-14,304 (1 microM) potentiated these responses (to 36 +/- 4, 67 +/- 7, and 135 +/- 17 mm Hg, respectively). This angiotensin II/alpha(2)-adrenoceptor interaction was abolished by U-73122 (10 microM), GF109203X (3 microM), CGP77675 (5 microM), and wortmannin (0.2 microM). Preglomerular microvascular smooth muscle cells expressed phospholipase (PLC)-beta(2), PLC-beta(3), c-src, phospho(tyrosine 416)-c-src, and PI3K. In these cells, angiotensin II (0.1 microM) and UK-14,304 (1 microM) per se did not increase phospho-c-src; however, the combination of angiotensin II plus UK-14,304 doubled phospho-c-src, and this interaction was abolished by U-73122 (10 microM) and GF109203X (3 microM). In conclusion, the PLC/PKC/c-src/PI3K pathway may contribute importantly to the interaction between alpha(2)-adrenoceptors and angiotensin II on renal vascular resistance.     

Effects of intracavernous administration of selective antagonists of alpha(1)-adrenoceptor subtypes on erection in anesthetized rats and dogs

The proerectile properties of three novel alpha(1)-adrenoceptor (alpha(1)-ADR) antagonists with different profiles of selectivity for the alpha(1)-ADR subtypes have been evaluated in anesthetized rats and dogs on intracavernous (IC) injection, in comparison with prazosin and phentolamine. In rats, the tested compounds decreased blood pressure (BP) and increased IC pressure (ICP), as well as the ratio ICP/BP. Rec 15/2841 (alpha(1a)- plus alpha(1L)-ADR-selective antagonist) and Rec 15/2615 (alpha(1b)-ADR selective) were the most potent compounds. The ICP/BP ratios calculated after injection of Rec 15/3039 (alpha(1d)-ADR selective) were not markedly different from those observed after vehicle injection. Prazosin and phentolamine proved poorly active, their main effect being hypotension. Approximate ED(25) values (dose of compound in micrograms inducing 25% increase of ICP/BP ratio) were Rec 15/2615 (22 microgram/kg)>= Rec 15/2841 (29 microgram/kg) > prazosin (136 microgram/kg) > phentolamine (1298 microgram/kg) > Rec 15/3039 (9600 microgram/kg). Submaximal stimulation of the cavernous nerve elicited an ICP rise whose amplitude was not altered by Rec compounds. In contrast, prazosin and phentolamine decreased this ICP rise. All compounds but 15/3039 induced significant increase of the ICP/BP ratio in dogs. Rec 15/2615 proved to be the most interesting compound, inducing significant increases of ICP/BP at doses practically devoid of effects on BP. The rank order of potency in dog in increasing the ICP/BP ratio was similar to that observed in rats. Only at the highest doses tested, all compounds, except Rec 15/3039, decreased the ICP rise elicited by submaximal stimulation of the cavernous nerve. Our data demonstrate that the alpha(1b)- and alpha(1L)-ADR subtypes are functionally relevant for the erectile function in these models, and that alpha(1b)- and/or alpha(1L)-ADR subtypes selective antagonists could represent a real advantage in erectile dysfunction therapy.     

Bidirectional allosteric effects of agonists and GTP at alpha(2A/D)-adrenoceptors

Agonists and GTP exert reciprocal effects on the stability of the G protein-coupled receptor/G protein complex, implying bidirectional control over the receptor/G protein interface. To investigate this relationship, we compared the ability of a series of hydroxyl-substituted phenethylamine and imidazoline agonists to stimulate [(35)S]guanosine 5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) binding in membranes from alpha(2A/D)-adrenergic receptor-transfected PC12 cells with the magnitude of the GTP-induced reduction in agonist affinity in [(3)H]rauwolscine-binding studies. Agents previously described as full and partial agonists in functional studies showed similar relative efficacies in promoting GTP binding (r = 0.97) as well as similar relative potencies (r = 0.94). Efficacy among agonists for promotion of [(35)S]GTPgammaS binding was closely correlated with the relative influence of GTPgammaS on agonist binding (r = 0.97), consistent with a bidirectional allosteric influence by agonists and GTP on receptor/G protein complexation. In an additional series of tolazoline derivatives, a range in efficacy from full agonism to strong inverse agonism was observed, depending on the presence or absence of hydroxyl substituents. Together these results suggest that agonist-induced repositioning of transmembrane helices via their hydroxyl interactions is a critical determinant of the stability of the receptor/G protein complex and therefore of agonist efficacy.     

Characterization of the alpha(2)-adrenoceptor subtype, which functions as alpha(2)-autoreceptor in human neocortex

The pharmacological properties of the alpha(2)-adrenergic receptors regulating the release of norepinephrine were investigated in human neocortex. Slices were preincubated with [(3)H]norepinephrine, superfused under blockade of transmitter reuptake, and stimulated electrically. First, the autoinhibitory circuit of [(3)H]norepinephrine release was analyzed quantitatively by estimation of the K(d) of norepinephrine at the alpha(2)-autoreceptor (10(-7.99) M), the concentration of the endogenous transmitter causing this autoinhibition at a stimulation frequency of 3 Hz (10(-7.61) M), and the maximum inhibition obtainable through the autoreceptor (83%). Second, antagonist pK(b) values of nine antagonists were determined by using their pEC(50) values (negative logarithms of antagonist concentrations that increased the electrically evoked overflow of tritium by 50%) against the release-inhibiting effect of the endogenous transmitter. When compared with binding or functional data from the literature, the pK(b) values correlated best with the antagonist affinities at alpha(2A) binding sites. In contrast, the correlations with alpha(2B), alpha(2C), and alpha(2D) sites were not as good. It is concluded that in human neocortex prejunctional autoreceptors are alpha(2A).     

Undifferentiated effects of calcium antagonists on pressor responses to selective alpha-1 and alpha-2 adrenoceptor agonists in anesthetized, spinal dogs

Whether pressor responses mediated by alpha-2 adrenoceptors are more susceptible to calcium antagonists than those mediated by alpha-1 adrenoceptors was investigated in anesthetized, spinal dogs. All drugs were administered i.v. Methoxamine (3-100 mu/kg) or xylazine (3-300 micrograms/kg) produced a sustained increase in mean arterial pressure but almost no effect on heart rate. Both the initial and the sustained phase of the pressor response to methoxamine were selectively antagonized by prazosin, whereas those to xylazine were selectively antagonized by yohimbine. These results indicate that the peripheral arterial bed of the dog comprises alpha-1 and alpha-2 adrenoceptors and that both the initial and sustained phases of the pressor response to methoxamine are predominantly mediated by alpha-1 adrenoceptors, whereas those to xylazine are mediated by alpha-2 adrenoceptors. The calcium antagonists, i.e., nifedipine (0.3-3 micrograms/kg), diltiazem (10-100 micrograms/kg) and KB-944 (10-100 micrograms/kg), administered during the sustained phase of the pressor responses to equieffective doses of methoxamine (100 micrograms/kg) and xylazine (1000 micrograms/kg), lowered mean arterial pressure. The three calcium antagonists in these doses also lowered the baseline mean arterial pressure but to a lesser extent than the elevated one. These results altogether indicate that the calcium antagonists were more effective in lowering mean arterial pressure elevated by either an alpha-1 or alpha-2 adrenoceptor agonist than the base-line mean arterial pressure and that the sustained phase of the pressor response mediated by alpha-1 adrenoceptors would involve Ca++ influx as much as or more than those mediated by alpha-2 adrenoceptors.     

Localization of sites in periventricular forebrain mediating cardiovascular effects of gamma-aminobutyric acid agonists and antagonists in anesthetized cats

Previous studies have shown that injection of gamma-aminobutyric acid (GABA) antagonists such as bicuculline methiodide (BMI) into the forebrain (i.e., lateral and third) ventricular system elicits neurally mediated increases in blood pressure and heart rate and inhibits baroreflex bradycardia in anesthetized cats. Similar administration of muscimol, a GABA agonist, blocks or reverses the effects of BMI but has no effect on blood pressure or heart rate in untreated animals. These findings suggest that GABAergic inhibition may tonically suppress a forebrain mechanism capable of modifying autonomic outflow to the cardiovascular system. In the present study, we used a technique designed to restrict the distribution of intraventricularly administered drugs to varying degrees in order to better localize the relevant sites of drug action. Our findings show that BMI increases heart rate and blood pressure and that muscimol counters these changes by acting at a site that is accessible from the intermediate (as opposed to the rostral or caudal) region of the third ventricle. In contrast, these agents influence baroreflex bradycardia by acting at more rostral periventricular sites. These findings are consistent with the notion that the GABAergic mechanisms involved in the cardiovascular effects resulting from intraventricular administration of BMI and muscimol are located in the periventricular hypothalamus.     

Regulation of aquaporin-2 expression by the alpha(2)-adrenoceptor agonist clonidine in the rat.

Aquaporin-2 (AQP-2), the major water channel responsible for water balance, has been shown to be regulated by the binding of vasopressin to V(2) vasopressin receptors in the medullary collecting duct. alpha(2)-Adrenoceptor agonists such as clonidine have been associated with an increase in free water clearance that was secondary to an inhibition of the ability of vasopressin to increase cAMP levels in the collecting ducts. This investigation focused on the possibility that this increase in free water clearance following administration of an alpha(2)-adrenoceptor agonist was associated with a reduction in medullary AQP-2 expression. In the anesthetized rat, clonidine increased urine flow rate (32+/-5 versus 137+/-16 microl/min, p<.05) and free water clearance (-58+/-6 versus 3+/-8 microl/min, p<.05) compared with the group receiving the saline vehicle infusion. The increase in free water clearance with clonidine administration was associated with a reduction in whole kidney AQP-2 mRNA levels (282+/-25 versus 216+/-11 A units, p<.05). This decrease in water reabsorption was associated with a redistribution of AQP-2 away from the luminal membrane of the medullary collecting duct to the cytosol. These effects were not secondary to changes in serum vasopressin levels, as these were similar in the vehicle control and clonidine groups (59+/-5 pg/ml versus 64+/-7 pg/ml, p = NS). The rapid redistribution of AQP-2 and the reduction in AQP-2 mRNA following clonidine administration are consistent with the hypothesis that the alpha(2) adrenoceptor regulates water excretion at least in part by effects on AQP-2.