Alpha 2-adrenoceptor Antagonist Research Articles

Inhibition of human prostate cancer cells proliferation by a selective alpha1-adrenoceptor antagonist labedipinedilol-A involves cell cycle arrest and apoptosis

In this research, we conducted an in vitro analysis to evaluate the prostate cancer cells response to labedipinedilol-A in order to determine the effect of this selective α 1-adrenoceptor antagonist to suppress prostate cancer cell growth by affecting cell proliferation and apoptosis. Here, we report that treatment of androgen-sensitive (LNCaP) and androgen-insensitive (PC-3) prostate cancer cells with labedipinedilol-A inhibited cell proliferation in concentration-dependent and time-dependent manners. Moreover, norepinephrine-stimulated proliferation of both cell lines are markedly inhibited by labedipinedilol-A. The probable involvement of α 1-adrenoceptors in this cellular response is suggested. Labedipinedilol-A-induced growth inhibition was associated with G 0/G 1 arrest, and G 2/M arrest depending upon concentrations. Cell cycle blockade was associated with reduced amounts of cyclin D1/2, cyclin E, Cdk2, Cdk4, and Cdk6 and increased levels of the Cdk inhibitory proteins (Cip1/p21 and Kip1/p27). In addition, labedipinedilol-A also induced apoptosis in PC-3 cells, as determined by using Hoechst 33342 staining, DNA fragmentation, and Annexin V staining assay. Furthermore, labedipinedilol-A triggered the mitochondrial apoptotic pathway, as indicated by increasing the expression of Bax, but decreasing the level of Bcl-2, resulting in mitochondrial membrane potential loss, cytochrome c release, and activation of caspase-9 and -3. We further investigated the role of MAPK cascades in the anti-proliferative and apoptosis effects of labedipinedilol-A, and confirmed that labedipinedilol-A could activate JNK1/2 but not p38 in both cell lines. Unlike JNK1/2, however, labedipinedilol-A treatment resulted in down-regulation of phospho-ERK1/2 expression. We concluded that labedipinedilol-A possessed the growth-suppressive and apoptotic effects on LNCaP and PC-3 cells by its α 1-adrenoceptor blockade, and the apoptotic effects of labedipinedilol-A primarily through caspases and MAPKs mediated pathways.

Effects of Long-Term Dietary Soy Treatment on Female Urethral Morphology and Function in Ovariectomized Nonhuman Primates

Agonistic effects of estrogen on the female urethra include an increase in contractile function, blood flow and mucosal hyperplasia. Whether such effects can be achieved by soy based phytoestrogen diets is unclear. We studied the effects of chronic phytoestrogen treatment on the structural and functional properties of the urethra in ovariectomized monkeys. Following ovariectomy 18 monkeys were fed a diet containing soy (9) or casein (9) based protein for 32 months. At necropsy the urethra and bladder were removed and the urethra was separated into 3 segments of equal length, including a proximal, a middle and a distal segment. Each urethral segment and 1 bladder segment was tested in vitro for functional responses to electrical field stimulation and pharmacological stimulation, and the proximal to distal segments were tested for urothelial thickness and mucosal area. Electrical field stimulation produced frequency dependent contractile responses in the bladder, proximal and middle segments but not in the distal segment. Carbachol, phenylephrine and endothelin-1 produced concentration dependent contractions in all urethral segments. The maximum response decreased from the proximal to the distal segment (p </=0.05). The maximum response in the proximal and bladder segments to pharmacological stimulation and electrical field stimulation as well as mucosal area and urothelial thickness were significantly higher in the treatment than in the control group (p < or =0.05). Chronic phytoestrogen treatment results in increased responsiveness of the proximal urethra to pharmacological and electrical stimulation, which is associated with an increase in urothelial thickness and mucosal area. This study indicates that dietary soy may have estrogen agonist effects on the urethra and bladder in estrogen deficient females.

THE EFFECT OF DOBUTAMINE ON PLATELET AGGREGATORY FUNCTION IN NEWBORN PIGLETS WITH HYPOXIA AND REOXYGENATION

Dobutamine, a beta-adrenoceptor agonist that is often used to treat myocardial dysfunction in asphyxiated neonates, may act on the adrenoceptors of platelets resulting in activation. Little information is available on the effect and mechanistic pathway of dobutamine on the platelet aggregatory function in neonatal asphyxia. Newborn piglets were acutely instrumented and exposed to hypoxia for 2 h and reoxygenation for 4 h. Piglets were randomized to receive dobutamine infusion (5, 10, or 20 microg/kg per min) or saline (hypoxic-control) at 2 to 4 h of reoxygenation (n = 8 each), and sham-operated animals were not exposed to hypoxia and reoxygenation (n = 6). Platelet number, collagen-stimulated whole blood aggregation, and plasma concentrations of thromboxane B2 were studied. The effects of alpha- and beta-adrenoceptor antagonists (phentolamine and propranolol, respectively) on platelet aggregation to in vitro administration of dobutamine (3 microM) were also examined. Shock and metabolic acidosis developed similarly in all hypoxia-reoxygenated groups. At 4 h of reoxygenation, platelet numbers in all groups decreased, with no differences among groups. Platelet aggregation deteriorated significantly with a rightward shift of concentration-response curve in piglets receiving 10 and 20 microg/kg per min of dobutamine. The group that received 20 microg/kg per min of dobutamine had increased plasma thromboxane B2 concentrations from baseline (P < 0.05). The platelet aggregatory response induced by 3 microM of dobutamine was improved by the coadministration of the beta-but not the alpha-adrenoceptor antagonist. We observed platelet aggregatory dysfunction in hypoxic-reoxygenated newborn piglets treated with high-dose dobutamine. Further investigation is needed to examine the differential effects of dobutamine and hypoxia-reoxygenation in platelet aggregation in newborns.

The Antiallodynic Effect of Neurotropin® Is Mediated via Activation of Descending Pain Inhibitory Systems in Rats with Spinal Nerve Ligation

Neurotropin, a nonprotein extract isolated from inflamed skin of rabbits inoculated with vaccinia virus, is widely used in Japan to treat chronic pain such as neuropathic pain. Although some studies have been conducted on the mechanism of the antiallodynic action of Neurotropin, this mechanism has yet to be adequately clarified. The left fifth lumbar nerve of rats was tightly ligated with silk sutures under pentobarbital anesthesia. Mechanical allodynia was confirmed by measuring the hindpaw withdrawal threshold in response to application of von Frey filaments. Behavioral tests were performed at 28 days after nerve ligation. Neurotropin was administered IV, intrathecally or intracerebroventricularly in L5 spinal nerve ligation (L5-SNL) rats. We examined the effects of noradrenergic, serotonergic and gamma-aminobutyric acid (GABA)ergic antagonists on the antiallodynic action of Neurotropin in L5-SNL rats. Yohimbine hydrochloride (yohimbine) was used as an alpha(2) adrenoceptor antagonist, ketanserin tartrate (ketanserin) as a 5-HT(2A) receptor antagonist, MDL72,222 as a 5-HT(3) receptor antagonist, (-)-bicuculline methobromide (bicuculline) as a GABA(A) receptor antagonist, and CGP35,348 as a GABA(B) receptor antagonist, and intrathecally injected. IV (50-100 NU/kg) doses of Neurotropin elicited an antiallodynic action in L5-SNL rats. Moreover, intracerebroventricular (400 mNU/rat), but not intrathecal, injection of Neurotropin inhibited allodynia. The antiallodynic action of Neurotropin (100 NU/kg, IV) was antagonized by intrathecal injections of yohimbine (10 nmol/rat), ketanserin (30 nmol/rat), MDL72,222(30 nmol/rat), bicuculline (0.6 nmol/rat) and CGP35348 (30 nmol/rat). On the other hand, the antiallodynic action of intrathecally injected m-CPBG (5-HT(3) receptor agonist) was reversed by intrathecal injection of bicuculline and CGP35348, suggesting interaction of 5-HT(3) receptors and spinal inhibitory (GABAergic) interneurons. These results suggest that the antiallodynic effect of Neurotropin is mediated via activation of descending pain inhibitory systems, such as the noradrenergic and serotonergic systems, which project from supraspinal sites to the spinal dorsal horn. In addition, activation of inhibitory GABAergic interneurons via 5-HT(3) receptors by serotonin released in the spinal dorsal horn may also be involved in the antiallodynic action of Neurotropin.

Inhibition of Seminal Emission Is the Main Cause of Anejaculation Induced by a New Highly Selective α1A-Blocker in Normal Volunteers

Recent studies have highlighted the influence of alpha1-adrenoceptor antagonists on ejaculatory function. We evaluated the effect of a new, highly selective alpha1A-blocker, silodosin, on ejaculatory function of normal volunteers. The study included 15 healthy male urologists who voluntarily participated in the study. They took 4 mg of silodosin or a placebo twice daily for 3 days in a randomized, double-blind crossover design. We investigated the ejaculatory volume, sperm count in urine after ejaculation, and fructose concentration in seminal plasma before and after administration of the agents. All volunteers on silodosin had a complete lack of ejaculation. Three days after completion of silodosin, the mean ejaculatory volume recovered to the baseline level. There was no sperm in urine after ejaculation under silodosin administration in any volunteer. All volunteers on silodosin had anejaculation and did not show post-ejaculate sperm in their urine. The mechanism of ejaculatory dysfunction caused by silodosin is a loss of seminal emission.

Risperidone, an atypical antipsychotic enhances the antidepressant-like effect of venlafaxine or fluoxetine: Possible involvement of alpha-2 adrenergic receptors

Clinical studies have reported the beneficial outcome of addition of lower doses of risperidone to antidepressant therapy specifically with selective serotonin reuptake inhibitors (SSRIs) in the treatment of major depression. The present study, therefore, examined the beneficial effect, if any, of addition of risperidone (an atypical antipsychotic) to the antidepressant-like effect of venlafaxine (dual reuptake inhibitors of both serotonin and norepinephrine, SNRI) or fluoxetine (SSRI) in Porsolt's Forced Swim Test (FST) using male laca mice. Attempts have been made to study the involvement of alpha-2 adrenergic receptors in the mechanism of action. Immobility period was recorded for a period of 6 min. Venlafaxine (4 and 8 mg/kg, i.p.) or fluoxetine (10 and 20 mg/kg, i.p.) inhibited the immobility period in mice. Addition of risperidone (0.1 mg/kg, i.p.) potentiated the anti-immobility effect of either venlafaxine (4 and 8 mg/kg, i.p.) or fluoxetine (10 and 20 mg/kg, i.p.) in mouse FST. Furthermore, the anti-immobility effect of combination of risperidone (0.1 mg/kg, i.p) plus venlafaxine (4 mg/kg, i.p.) or fluoxetine (10 mg/kg, i.p.) was potentiated by the addition of yohimbine (2 mg/kg, i.p.), an alpha-2 adrenoceptors antagonist. The results of the present study suggest that the beneficial consequences of addition of risperidone with venlafaxine or fluoxetine in mouse forced swim test may involve alpha-2 adrenergic receptors.

Effects of pharmacological stressors on c-fos and CRF mRNA in mouse brain: Relationship to alcohol seeking

A marked heterogeneity exists among stressors in their ability to reinstate alcohol seeking in rats. We have reported that the pharmacological stressor yohimbine, an alpha-2 adrenoceptor antagonist, potently reinstated alcohol seeking, but FG-7142, a benzodiazepine inverse agonist was ineffective. In rats, we determined that yohimbine elicits patterns of brain expression of the mRNAs for c-fos, a marker of neuronal activation, and corticotropin-releasing factor (CRF) a stress-related peptide, distinct from that produced by FG-7142. The purpose of the present experiment is to determine if these differential effects of yohimbine and FG-7142 on regional c-fos and CRF mRNA expression generalize to another animal commonly used in alcohol research, the C57 BL/6J mouse. In comparing the results of the present study to those of our previous one, we found a number of commonalities in the patterns of activation elicited by yohimbine and FG-7142 between the two species, and some notable differences. As we found in the rat, yohimbine selectively increased c-fos mRNA in the mouse NACs, BLA and CeA. Yohimbine increased CRF mRNA only in the mouse PVN, but was without effect on CRF mRNA in extrahypothalamic sites, the BNST and CeA. This differs from what we saw in the rat, where yohimbine increased CRF mRNA in these extrahypothalamic regions, but not the PVN. The selective induction of c-fos in the NACs, BLA and CeA of mice and rats by yohimbine offers further support for the idea that activation of these structures participates in reinstatement induced by such stressors.

Intrathecal Clonidine Suppresses Phosphorylation of the N-Methyl-D-Aspartate Receptor NR1 Subunit in Spinal Dorsal Horn Neurons of Rats with Neuropathic Pain

Intrathecal (IT) administration of the alpha-2 adrenoceptor agonist, clonidine, produces significant analgesic effects. Although several mechanisms underlying clonidine-induced analgesia have been proposed, the possible interaction with N-methyl-D-aspartate (NMDA) receptors as a major antinociceptive mechanism has not been addressed. We designed the present study to determine whether clonidine or other analgesics can affect spinal NMDA receptor activation in rats with chronic constriction injury (CCI)-induced neuropathy. Rats underwent unilateral CCI, and received IT clonidine (1, 5, 20 microg/rat), [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO, mu opioid receptor agonist, 1 microg/rat), gabapentin (anticonvulsant, 100 microg/rat) or vehicle 2 wks later. After drug injection, we measured the pain response to thermal or mechanical stimuli and used immunohistochemistry to evaluate spinal cord phosphorylated NMDA-receptor subunit 1 (pNR1) expression. Two weeks after CCI surgery, rats displayed significant mechanical allodynia and thermal hyperalgesia, and the spinal cord dorsal horn showed a significant increase in the number of pNR1 immunoreactive neurons. IT injection of clonidine (20 microg/rat), DAMGO and gabapentin potently reduced mechanical allodynia and thermal hyperalgesia. Importantly, IT clonidine, but not IT DAMGO or gabapentin, dose-dependently reduced CCI-induced pNR1 expression in all lamina of the spinal cord dorsal horn by 30 min after injection. In addition, IT injection of the alpha-2 adrenoceptor antagonist, idazoxan (40 microg/rat) 10 min before clonidine injection completely reversed clonidine's antihyperalgesic and antiallodynic effects, as well as clonidine's suppressive effect on CCI-induced NR1 phosphorylation in the spinal cord dorsal horn. Our data indicate that IT clonidine's antihyperalgesic/antiallodynic effect on neuropathic pain is associated with a significant reduction in spinal NMDA receptor phosphorylation and suggests a potentially novel mechanism of clonidine's action.

α2A‐Adrenoceptor antagonism increases insulin secretion and synergistically augments the insulinotropic effect of glibenclamide in mice

The imidazoline-type alpha2-adrenoceptor antagonists (+/-)-efaroxan and phentolamine increase insulin secretion and reduce blood glucose levels. It is not known whether they act by antagonizing pancreatic beta-cell alpha2-adrenoceptors or by alpha2-adrenoceptor-independent mechanisms. Many imidazolines inhibit the pancreatic beta-cell KATP channel, which is the molecular target of sulphonylurea drugs used in the treatment of type II diabetes. To investigate the mechanisms of action of (+/-)-efaroxan and phentolamine, alpha2A-adrenoceptor knockout (alpha2A-KO) mice were used. Effects of (+/-)-efaroxan, 5 mg kg(-1), and phentolamine, 1 mg kg(-1), on blood glucose and insulin levels were compared with those of the non-imidazoline alpha2-adrenoceptor antagonist [8aR,12aS,13aS]-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine (RS79948-197), 1 mg kg(-1), and the sulphonylurea glibenclamide, in alpha2A-KO and control (wild type (WT)) mice. In fed WT mice, (+/-)-efaroxan, phentolamine and RS79948-197 reduced blood glucose and increased insulin levels. Fasting abolished these effects. In fed alpha2A-KO mice, (+/-)-efaroxan, phentolamine and RS79948-197 did not alter blood glucose or insulin levels, and in fasted alpha2A-KO mice, blood glucose levels were increased. Glibenclamide, at a dose only moderately efficacious in WT mice (5 mg kg(-1)), caused severe hyperinsulinaemia and hypoglycaemia in alpha2A-KO mice. This was mimicked in WT mice by co-administration of RS79948-197 with glibenclamide. These results suggest that (+/-)-efaroxan and phentolamine increase insulin secretion by inhibition of beta-cell alpha2A-adrenoceptors, and demonstrate a critical role for alpha2A-adrenoceptors in limiting sulphonylurea-induced hyperinsulinaemia and hypoglycaemia.

Dexmedetomidine Enhances the Local Anesthetic Action of Lidocaine via an α-2A Adrenoceptor

Clonidine, an alpha-2 adrenoceptor agonist, is a common adjunct in both central and peripheral blocks. Dexmedetomidine, a more selective alpha-2 adrenoceptor agonist, is also known to enhance central neural blockades. Its peripheral effect, however, has not been fully elucidated. Thus, we evaluated the effect of dexmedetomidine and other alpha-2 adrenoceptor agonists on the local anesthetic action of lidocaine at the periphery and explored the mechanism involved. alpha-2 Adrenoceptor agonists, including dexmedetomidine, clonidine, and oxymetazoline, combined with lidocaine were intracutaneously injected into the back of male guinea pigs. The test of six pinpricks was applied every 5 min until 60 min after the injection. The number of times which the prick failed to elicit a response during the 60-min period was added and the sum served as an anesthetic score indicating the degree of local anesthesia. Differences from the control value within the group were analyzed using an analysis of variance followed by a post hoc Dunnett's test. Furthermore, we evaluated the antagonism of the effect of dexmedetomidine by yohimbine, an alpha-2A, 2B, and 2C adrenoceptor antagonist, or prazosin, an alpha-1, alpha-2B, and 2C adrenoceptor antagonist, analyzed using a two-way analysis of variance. All alpha-2 adrenoceptor agonists enhanced the degree of local anesthesia of lidocaine in a dose-dependent manner. Furthermore, yohimbine inhibited the effect of dexmedetomidine, whereas prazosin did not. We demonstrated that alpha-2 adrenoceptor agonists enhanced the local anesthetic action of lidocaine, and suggest that dexmedetomidine acts via alpha-2A adrenoceptors.

Differential Effects of α2-adrenoceptors in the Modulation of the Thermoregulatory Response in Mice Induced by Meperidine

Meperidine proved to be more effective in treatment of shivering than equianalgesic doses of other opioids, especially pure mu-agonists. Further, meperidine has well known nonopioid actions including agonistic effects at alpha2-adrenoceptors in vitro. Accordingly, the authors investigated nonopioid receptor-mediated effects of meperidine on thermoregulation using a mice model of nonshivering thermogenesis. To differentiate conceivable alpha2-adrenoceptor subtype specific interactions the authors analyzed wild-type mice and knock-out mice with deletion of the alpha2A-, alpha2B-, or alpha2C-adrenoceptor. Ten mice per group (n = 60) were injected with saline, meperidine (20 mg/kg), saline plus naloxone (125 microg/kg), meperidine plus naloxone, fentanyl (50 microg/kg) plus naloxone, or meperidine plus atipamezole (2 mg/kg) intraperitoneally. Each mouse was subjected to the six different treatments. Then they were positioned into a plexiglas chamber where rectal temperature and mixed expired carbon dioxide were measured while whole body cooling was performed. Maximum response intensity and thermoregulatory threshold temperature of nonshivering thermogenesis were analyzed. Meperidine decreased the thermoregulatory threshold temperature in wild-type mice and alpha2B- and alpha2C-adrenoceptor knock-out mice. This effect ended after injection of the alpha2-adrenoceptor antagonist atipamezole. In wild-type and alpha2B-adrenoceptor knock-out mice, the decrease of thermoregulatory threshold was not reversible by administration of the opioid receptor antagonist naloxone. In contrast, in alpha2A-adrenoceptor knock-out mice, no decline of thermoregulatory threshold following meperidine injection was detectable. Maximum response intensity of nonshivering thermogenesis was comparable in all groups. The authors' results suggest a major role of alpha2-adrenoceptors, especially the alpha2A subtype, in the mediation of thermoregulatory effects caused by meperidine in mice.

Phenoxybenzamine treatment can lead to loss of endothelial cell viability

Phenoxybenzamine, an irreversible alpha-adrenoceptor antagonist, is used as a topical treatment against catecholamine-induced contraction in radial artery bypass grafts. Published data suggest that a wide range of phenoxybenzamine doses may be equally effective. This study aimed to investigate whether lower doses of phenoxybenzamine would benefit grafts by better preserving endothelium. To this end human vascular endothelial cells were isolated from sections of radial artery or saphenous vein, and treated with phenoxybenzamine for 30 min. Cells were then washed free of drug and viability assayed using a resazurin-based toxicology assay or returned to culture for assay at 24 h. Phenoxybenzamine treatment showed a dose-dependent effect on cell viability over several clinically employed concentrations. Concentrations above 0.1 mM led to a loss of viability, which became more pronounced with time. The loss of viability was shown to be independent of the carrier used, as results were identical when phenoxybenzamine was dissolved in dimethylsulphoxide, which alone did not affect viability. Changes in pH alone were also not sufficient to affect viability. In conclusion, phenoxybenzamine treatment is likely to cause damage to graft endothelium if employed at concentrations above 0.1 mM (0.03 mg/ml). Phenoxybenzamine may be safely used at lower doses with no potential loss of endothelial cell viability.

Mechanisms of analgesic action of pulsed radiofrequency on adjuvant-induced pain in the rat: Roles of descending adrenergic and serotonergic systems

Pulsed radiofrequency (PRF) has been reported to be effective in the treatment of several types of pain. The mechanism of action, however, is not well known. In a recent study, the antinociceptive effects of acute thermal pain were shown to be mediated via descending pain inhibitory pathways. In this study we observed an analgesic effect of PRF treatment in an adjuvant induced inflammatory pain model in rats. In this model, sciatic nerves were treated with PRF at 37° and 42°, which inhibited hyperalgesia in the inflammatory groups when compared to RF and sham treatment. This effect was attenuated after intrathecal administration of the alpha2-adrenoceptor antagonist yohimbine, the selective 5-HT3 serotonin receptor antagonist MDL72222, and the non-selective serotonin receptor antagonist methysergide. All three drugs were found to significantly inhibit the analgesic effect of PRF. The results suggest that the analgesic action of PRF involves the enhancement of noradrenergic and serotonergic descending pain inhibitory pathways.

Cellular mechanism of adrenalin stimulated chloride secretion via beta‐adrenoceptor in T84 cells

In the present study, the intracellular regulatory pathways involved in the adrenalin-stimulated chloride secretion across T84 cells were investigated. Biphasic characteristics were observed in the Isc response to the basolateral addition of adrenalin (0.25 nM-100 microM). The biphasic response was almost abolished by removing ambient Cl(-). Chloride secretion was found to depend on the activities of basolaterally located Na+-K+-2Cl(-) cotransporters and K+ channels. The alpha-adrenoceptor antagonist phentolamine did not have any effect on either phase of adrenalin-induced Isc, while after pretreatment of the beta-adrenoceptor antagonist propranolol, the adrenalin-induced Isc was substantially abolished, suggesting the biphasic response may be mediated by the beta-adrenoceptor. Under whole cell patch-clamp conditions, T84 cells responded to adrenalin with a rise in inward current. The current, which exhibited a linear I-V relationship and time- and voltage-independent characteristics, was inhibited by the chloride channel blocker DPC and the reverse potential was close to the equilibrium potential for Cl(-) (0 mV), implying that the current was Cl(-) selective. When preloaded with a Ca2+-chelating agent, BAPTA/AM did not affect the Isc response to adrenalin, whereas the Isc was destroyed by pretreating the cells with an adenyl cyclase inhibitor, MDL12330A. These observations were further supported by the intracellular [cAMP] measurement experiment, indicating that adrenalin induced chloride secretion could be mediated by a beta-adrenoceptor only involving cAMP as an intracellular second messenger.

Guanidine and 2-Aminoimidazoline Aromatic Derivatives as α2-Adrenoceptor Antagonists. 2. Exploring Alkyl Linkers for New Antidepressants

The preparation of a number of (bis)guanidine and (bis)2-aminoimidazoline derivatives as potential alpha 2-adrenoceptor antagonists for the treatment of depression is presented. Human brain tissue was used to measure their affinity toward the alpha 2-adrenoceptors in vitro. Compounds 6b, 8b, 9b, 10b, 15b, 17b, 18b, 20b, and 21b displayed a good affinity (pKi > 7) and were evaluated in in vitro functional [(35)S]GTPgammaS binding assays in human prefrontal cortex to determine their agonistic or antagonistic activity. Among these compounds, 17b and 20b showed the expected behavior for an antagonist and were subject to in vivo microdialysis experiments in rats. Significantly, these experiments confirmed the antagonistic properties of 17b and 20b, and therefore both compounds can be considered as potential antidepressants.

Selective ablation of dorsal horn NK 1 expressing cells reveals a modulation of spinal alpha2-adrenergic inhibition of dorsal horn neurones

Activity in descending systems from the brainstem modulates nociceptive transmission through the dorsal horn. Intrathecal injection of the neurotoxin saporin conjugated to SP (SP–SAP) into the lumbar spinal cord results in the selective ablation of NK 1 receptor expressing (NK 1+ve) neurones in the superficial dorsal horn (lamina I/III). Loss of these NK 1+ve neurones attenuates excitability of deep dorsal horn neurones due to a disruption of both intrinsic spinal circuits and a spino-bulbo-spinal loop, which activates a descending excitatory drive, mediated through spinal 5HT 3 receptors. Descending inhibitory pathways also modulate spinal activity and hence control the level of nociceptive transmission relayed to higher centres. To ascertain the spinal origins of the major descending noradrenergic inhibitory pathway we studied the effects of a selective alpha2-adrenoceptor antagonist, atipamezole, on neuronal activity in animals pre-treated with SP–SAP. Intrathecal application of atipamezole dose dependently facilitated the mechanically evoked neuronal responses of deep dorsal horn neurones to low intensity von Frey hairs (5–15 g) and noxious thermal (45–50 °C) evoked responses in SAP control animals indicating a physiological alpha2-adrenoceptor control. This facilitatory effect of atipamezole was lost in the SP–SAP treated group. These data suggest that activity within noradrenergic pathways have a dependence on dorsal horn NK 1+ve cells. Further, noradrenergic descending inhibition may in part be driven by lamina I/III (NK 1+ve) cells, and mediated via spinal alpha2-adrenoceptor activation. Since the same neuronal population drives descending facilitation and inhibition, the reduced excitability of lamina V/VI WDR neurones seen after loss of these NK 1+ve neurones indicates a dominant role of descending facilitation.

Endothelial α1‐adrenoceptors regulate neo‐angiogenesis

Intact endothelium plays a pivotal role in post-ischaemic angiogenesis. It is a phenomenon finely tuned by activation and inhibition of several endothelial receptors. The presence of alpha(1)-adrenoceptors on the endothelium suggests that these receptors may participate in regenerative phenomena by regulating the responses of endothelial cells involved in neo-angiogenesis. We evaluated the expression of the subtypes of the alpha(1)-adrenoceptor in isolated endothelial cells harvested from Wistar-Kyoto (WKY) rats. We explored the possibility these alpha(1)-adrenoceptors may influence the pro-angiogenic phenotype of endothelial cells in vitro. In vivo, we used a model of hindlimb ischaemia in WKY rats, to assess the effects of alpha(1) adrenoceptor agonist or antagonist on angiogenesis in the ischaemic hindlimb by laser Doppler blood flow measurements, digital angiographies, hindlimb perfusion with dyed beads and histological evaluation. In vitro, pharmacological antagonism of alpha(1)-adrenoceptors in endothelial cells from WKY rats by doxazosin enhanced, while stimulation of these adrenoceptors with phenylephrine, inhibited endothelial cell proliferation and DNA synthesis, ERK and retinoblastoma protein (Rb) phosphorylation, cell migration and tubule formation. In vivo, we found increased alpha(1)-adrenoceptor density in the ischaemic hindlimb, compared to non-ischaemic hindlimb, suggesting an enhanced alpha(1)-adrenoceptor tone in the ischaemic tissue. Treatment with doxazosin (0.06 mg kg(-1) day(-1) for 14 days) did not alter systemic blood pressure but enhanced neo-angiogenesis in the ischaemic hindlimb, as measured by all our assays. Our findings support the hypothesis that the alpha(1)-adrenoceptors in endothelial cells provide a negative regulation of angiogenesis.

Identification of the alpha‐2 adrenoceptor subtype causing contraction of rat lateral tail vein

Constriction of veins plays an important role in thermoregulation. Norepinephrine from sympathetic nerves can activate alpha‐2 adrenoceptors in veins causing constriction and reduced heat loss in response to cooling. We determined the alpha‐2 adrenoceptor subtype causing contraction of the rat lateral tail vein studied in vitro. Tail veins were isolated, endothelium removed, and incubated in Krebs solution at 37 degrees C. Concentration‐response curves for contraction caused by the alpha‐2 adrenoceptor agonist UK 14304 were generated in the absence and presence of the alpha‐2 adrenoceptor antagonists RX 821002 (non‐subtype selective), BRL 44408 (alpha‐2A selective), ARC 239 (alpha‐2B/C selective) and MK 912 (alpha‐2C selective). UK 14304 caused concentration‐dependent contraction of the tail vein with an EC50 of 8.3 nM. The affinities (KB) of antagonists in blocking UK14304 contraction were RX 821002 (2.3 nM), BRL 44408 (64 nM), ARC 239 (199 nM) and MK 912 (0.06 nM). The affinity of MK 912 was nearly the same as its affinity for alpha‐2C adrenoceptors reported by others, and affinities for other antagonists were consistent with alpha‐2C adrenoceptors. Thus, alpha‐2C adrenoceptors mediate UK 14304 induced contraction of the rat lateral tail vein. The development of alpha‐2C selective agents may be useful to selectively regulate venous function in diseases associated with altered thermoregulation.

Expression of α 1-Adrenoceptor Subtype mRNA as a Predictor of the Efficacy of Subtype Selective α 1-Adrenoceptor Antagonists in the Management of Benign Prostatic Hyperplasia

We examined the correlation between the expression of alpha1-adrenoceptor subtype mRNA in the prostate and the clinical efficacy of subtype selective alpha1-adrenoceptor antagonists. We discuss the possibility of individualizing drug therapy in patients with benign prostatic hyperplasia. A total of 33 patients randomized to the tamsulosin group and 28 randomized to the naftopidil group were enrolled in this study. Each group of patients was administered 0.2 mg tamsulosin hydrochloride or 50 mg naftopidil daily for 12 weeks. Four prostate needle biopsy specimens were obtained from the transition zone to examine the expression of alpha-adrenoceptor subtypes. Specimens were stored at -80 C until used for TaqMan quantitative reverse transcriptase-polymerase chain reaction, which was performed after 12 weeks of treatment. Based on the results of quantitative reverse transcriptase-polymerase chain reaction the tamsulosin and naftopidil groups were grouped into alpha1a-adrenoceptor dominant (22 and 12 patients) and alpha1d-adrenoceptor dominant (11 and 16, respectively) subgroups. The efficacy of tamsulosin hydrochloride and naftopidil differed depending on the dominant expression of the alpha1-adrenoceptor subtype in the prostate. Tamsulosin hydrochloride was more effective in patients with dominant expression of the alpha1a-adrenoceptor subtype, whereas naftopidil was more effective in those with dominant expression of the alpha1d-adrenoceptor subtype. The expression level of alpha1-adrenoceptor subtype mRNA in the prostate could be a predictor of the efficacy of subtype selective alpha1-adrenoceptor antagonists in patients with benign prostatic hyperplasia. This result implies that genetic differences are responsible for the diverse responses to these drugs.

Doxazosin reduces cell proliferation and increases collagen fibers in rat prostatic lobes

We investigated the effects of doxazosin (Dox), an alpha-adrenoceptor antagonist used clinically for the treatment of benign prostatic hyperplasia (BPH), on the rat prostatic complex by assessing structural parameters, collagen fiber content, cell proliferation, and apoptosis. Adult Wistar rats were treated with Dox (25 mg/kg per day), and the ventral (VP), dorsolateral, and anterior prostate (AP) regions of the prostate complex were excised at 3, 7, and 30 days after treatment. At 24 h before being killed, the rats were injected once with 5-bromodeoxyuridine (BrdU; thymidine analog) to label mitotically active cells. The prostates were weighed and processed for histochemistry, morphometry-stereology, immunohistochemistry for BrdU, Western blotting for proliferating cell nuclear antigen (PCNA), and the TUNEL reaction for apoptosis. Dox-treated prostate lobes at day 3 presented increased weight, an enlarged ductal lumen, low cubical epithelial cells, reduced epithelial folds, and stretched smooth muscle cells. However, at day 30, the prostates exhibited a weight reduction of approximately 20% and an increased area of collagen and reticular fibers in the stromal space. Dox also reduced epithelial cell proliferation and increased apoptosis in the three prostatic lobes. Western blotting for PCNA confirmed the reduction of cell proliferation by Dox, with the AP and VP being more affected than the dorsal prostate. Thus, Dox treatment alters epithelial cell behavior and prostatic tissue mechanical demand, inducing tissue remodeling in which collagen fibers assume a major role.