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Objectives: To review the main findings of the Euralox 1 study – a multicentre, randomised, double-blind study conducted in 1008 healthy postmenopausal women allocated to raloxifene ( n = 495) or continuous combined estrogen–progestin therapy (ccEPT; n = 513) for 6 months – and provide an overview of the risks and benefits of raloxifene and ccEPT. Methods: A review is provided of previously published findings on uterine safety (bleeding rates and changes in endometrial thickness and uterine volume), gynaecological adjudication, cardiovascular risk (lipids, fibrinogen), adverse events, compliance, treatment satisfaction and quality of life. New data on biochemical markers of bone turnover (serum N -telopeptides and C-terminal telopeptides of type I collagen; NTX and CTX) assessed before and after 6 months’ treatment are presented. Results: Raloxifene caused less uterine bleeding than ccEPT and, unlike ccEPT, did not alter endometrial thickness or uterine volume. Serum CTX and NTX levels were reduced in both treatment groups, but the reduction was significantly greater with ccEPT. The two treatments had differential effects on lipids and fibrinogen levels; raloxifene had more favourable effects on serum HDL, the LDL/HDL ratio, and plasma fibrinogen. Raloxifene was associated with fewer adverse events or discontinuations, and this was associated with higher treatment satisfaction and better self-reported compliance. Conclusions: The clinical risk-benefit profile of raloxifene derived from the intermediate endpoints of this study suggests that it may be a better alternative to ccEPT for preventing long-term postmenopausal health risks in healthy postmenopausal women who are not suffering from vasomotor symptoms.

OBJECTIVE: To compare the incident rate of abnormal endometrial findings in postmenopausal women receiving treatment with either 60 mg of raloxifene or a continuous combined estrogen plus progestin therapy containing 2 mg of 17-estradiol plus 1 mg of norethisterone acetate for a duration of up to 12 months. METHODS: One thousand eight asymptomatic postmenopausal women with osteoporosis or cardiovascular risk factors with an endometrial thickness of less than 5 mm at baseline participated in this prospective, randomized, double-blind trial that lasted 6 months; 347 of these women also participated in a 6-month extension. Women with repeated bleeding or an increase in endometrial thickness to above 5 mm were subjected to saline-infused sonohysterography or hysteroscopy with biopsy. Sonographic, histologic, and clinical findings were adjudicated by a panel of 4 experts blinded with respect to patients’ treatments. All adjudicated patients were grouped into 15 diagnostic categories according to predefined criteria. RESULTS: Three hundred thirty-four women needed adjudication during the core phase, 73 (14.7%) of those taking raloxifene and 261 (50.9%) taking continuous combined estrogen plus progestin therapy (P < .001). Compared with raloxifene, women using continuous combined estrogen plus progestin therapy had significantly higher rates of benign endometrial proliferation (8.8 versus 1.2%, P < .001), endometrial polyps (4.3 versus 2.0%, P .048), and cystic atrophy (5.5 versus 1.2%, P < .001). CONCLUSION: Women using continuous combined estrogen plus progestin therapy more often have benign endometrial pathology and, in our study, more often required the protocol-specific gynecological follow-up assessments for safety reasons, as compared with those using raloxifene. These findings are of clinical relevance when choosing the most appropriate therapy for postmenopausal health risks such as osteoporosis. (Obstet Gynecol 2004;103:881‐91. © 2004 by The American College of Obstetricians and Gynecologists.) LEVEL OF EVIDENCE: I

As the olfactory system is closely connected with the brain areas responsible for the most crucial alterations in psychiatric populations, especially cognitive and emotional impairments, the study of olfactory processing may be a relevant approach to identify specific markers of alcohol dependence. The aim of this study was to propose the probable olfactory markers for alcohol dependence through a study of the olfactory parameters that involve the central olfactory pathway. We recruited the same 41 alcohol-dependent patients in an early (day 8) and late (day 67) stage of abstinence and 41 controls matched for gender, age, and smoking status. The participants underwent clinical assessments and several olfactory evaluations. The results revealed on one hand, the persistence of decreased intensity of positive emotion (happy), increased intensity of negative emotion (sad), and increased citation of surprise in patients, suggesting the presence of probable trait markers of alcohol dependence. On the other hand, we found decreased hedonic score for pleasant and neutral odorants, and decreased odor familiarity judgment only in the early stage of evaluation as probable state markers for alcohol dependence. These results may be underpinned by several neuropsychological alterations specific to this disease and their evolution after weaning. Further studies are needed to replicate these findings and to confirm the specificity and sensitivity of the olfactory tests in a larger sample of patients. The olfactory perception of all controls must be also retested in order to determine the specific state and/or trait markers of alcohol dependence.

LY 186641 is a diarylsulfonylurea with a broad spectrum antitumor activity against both murine and human solid tumors. We report here the results of a phase II trial of LY 186641 in advanced renal cell adenocarcinoma. The drug was administered orally, once daily for 2 weeks, every 21 days at a 700 mg/m2/d dose. Sixteen patients were enrolled in this phase II trial; 12 males, 4 females, with a median age of 58 years. All patients had progressive measurable metastatic disease. The primary tumor was surgically removed in all but one patient. Three patients were previously treated by biologic response modifiers (BRMs). A total of 72 courses were administered. The most common side effects were methemoglobulinemia (MetHgb) and anemia which occurred in 13 and 10 patients respectively. The MetHgb did not exceed 15%, and only 3 patients required blood transfusion for grade 3 (2 patients) and grade 4 (1 patient) anemia. Reversible hepatotoxicity was observed in 3 patients. There were one pathological complete response, seven stable disease and 8 progressive disease.

Unilateral microinjections of quipazine (0.9 μg in 50 nl) into the subretrofacial nucleus produced hypertension and a slight tachycardia associated with an increase in renal sympathetic nerve activity. Microinjections of quipazine lateral, caudal or rostral to this nucleus failed to alter blood pressure and heart rate. Similarly, microinjections of l-glutamate (3 nmol in 15 nl) into the subretrofacial nucleus elicited hypertension, tachycardia and renal sympatho-excitation. The magnitude of the pressor response to quipazine was smaller than the response elicited by l-glutamate but its duration was longer. Microinjections of quipazine into the lateral tegmental field at l-glutamate hypertensive sites failed to alter arterial blood pressure and heart rate. In contrast, microinjections of quipazine into the caudal ventrolateral medulla or into the nucleus tractus solitarii produced hypotension and sympatho-inhibition. These effects were prevented by microinjections of the 5-HT 2 receptor antagonists, LY 53857 or BW 501C. The present results indicate that stimulation of 5-HT 2 receptors of the subretrofacial nucleus produces hypertension and sympatho-excitation whereas stimulation of 5-HT 2 receptors in the caudal ventrolateral medulla and in the nucleus tractus solitarii produces hypotension and sympatho-inhibition.

Quipazine (0.5 mg/kg i.v.) produced a sustained pressor response and an increase in splanchnic nerve activity in intact as well as in baroreceptor-denervated cats without causing a significant change in heart rate. These effects were prevented by the 5-HT2 receptor antagonists, ritanserin (0.5 mg/kg i.v.) or BW 501 C (0.5 mg/kg i.v.). Quipazine induced an hypertensive response and an increase in splanchnic discharge in cats pretreated with prazosin (0.1 mg/kg) or hexamethonium (10 mg/kg i.v.). Bilateral application of quipazine (25 micrograms/side) to the ventrolateral pressor area produced a rapid increase in mean blood pressure and in splanchnic discharge. Pretreatment with prazosin (0.1 mg/kg i.v.) abolished the hypertension but not the sympatho-excitatory effects of quipazine. Local application of the 5-HT2 receptor antagonists, LY53857 (10 micrograms/side) or cyproheptadine (10 micrograms/side), had no effects on blood pressure and splanchnic nerve activity but prevented or reversed the actions of locally applied quipazine. LY 53857 (10 micrograms/side) antagonized the sympatho-excitatory effects of systemically administered quipazine. These results indicate that the cardiovascular changes induced by quipazine in anaesthetized cats are mediated by central 5-HT2 receptors located in the ventrolateral pressor area and by peripheral vascular 5-HT2 receptors.

Quipazine (0.5–2 mg/kg i.v.) produced transient hypotension and bradycardia followed by sustained hypertension and variable effects on heart rate in anaesthetized rats. The hypotension, bradycardia and sympatho-inhibitory effects of quipazine were attenuated by bivagotomy. In bivagotomized rats, the hypertension produced by quipazine was not modified by hexamethonium or prazosin but was abolished by ritanserin (1 mg/kg i.v.). In ritanserin-treated rats, section of the carotid sinus nerves and vagus nerves or ICS 205.930 (0.1 mg/kg i.v.) abolished the hypotensive, bradycardic and sympatho-inhibitory effects of quipazine; the action of quipazine was not reproducible in these rats. Quipazine also inhibited the Bezold-Jarish reflex elicited by 5-HT (20 μg/kg i.v.). In ICS 205.930-treated rats, the hypertension evoked by quipazine was associated with a reduction in splanchnic nerve activity due to stimulation of baroreceptors. The renin-angiotensin system is not involved in the hypertensive response. The increase in heart rate produced by quipazine in bivagotomized rats was reduced by ritanserin and tertatolol (0.1 mg/kg i.v.) and abolished by a combination of both drugs. We conclude that the bradycardic and sympatho-inhibitory effects of quipazine result from activation of 5-HT 3 receptors located in the cardiopulmonary area and of carotid body chemoreceptors. The hypertension and tachycardia are mediated by vascular and myocardial 5-HT 2 receptors. No evidence was obtained for a central sympatho-excitatory effect.

A pharmacological analysis of the effects of 5-HT on heart rate has been performed in the pithed rat. 5-HT induced a dose-dependent increase in heart rate whereas 5-HT1 receptor agonists--8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 5-methoxy-N,N-dimethyl-tryptamine (5-MeODMT), 5-methoxy 3-(1,2,3,6-tetrahydro-4-piridinyl) 1H indole (RU 24969) and 1-(m-trifluoromethylphenyl)-piperazine (TFMPP)--failed to increase heart rate. The increase in heart rate induced by the selective 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) was not significant. The dose-response curve to 5-HT for its tachycardic effects was shifted two-fold to the right by ketanserin and LY 53857 and nine-fold to the right by methiothepin. The effects of high doses of 5-HT (higher than 100 micrograms/kg iv) were antagonized by methiothepin, (-)propranolol, 2-(2-[4(O-methoxyphenyl)-piperazine-1-yl]-ethyl)4,4-dimethyl-1,3 (2H-4H) isoquinoline-dione (AR-C 239) and by pretreatment with reserpine. The 5-HT1 receptor antagonists, pindolol and spiroxatrine, the 5-HT3 receptor antagonist MDL 72222 and the alpha 2-adrenoceptor blocking agent idazoxan failed to antagonize the tachycardia induced by 5-HT. It is concluded that in the pithed rat, the tachycardia induced by 5-HT remained unexplained (implication of 5-HT2 receptors probably different from the classical vascular 5-HT2 receptor, or implication of 5-HT1C receptors?). Moreover, at high doses (higher than 100 micrograms/kg iv), 5-HT may increase heart rate by releasing catecholamines.

An attempt to determine the central site of the sympathoinhibitory effects of 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, has been made in normotensive anesthetized dogs. Intravenous and central (cisterna magna, vertebral artery) administration of 8-OH-DPAT induced dose-dependent decreases in blood pressure and sympathetic (splanchnic and renal) nerve activity in intact and baroreceptor denervated dogs. In contrast, 8-OH-DPAT was ineffective when injected into the nucleus tractus solitarii or into the raphe nuclei. Administration of 8-OH-DPAT into the ventrolateral pressor area (VLPA) decreased blood pressure and renal sympathetic nerve activity. The sympathoinhibitory effects of 8-OH-DPAT injected into the vertebral artery (v.a.) were antagonized by methiothepin and (+/-)pindolol injected into the v.a. Likewise, the effects of 8-OH-DPAT administered into the VLPA were antagonized by methiothepin injected into the same site. Depletion of central catecholamines and 5-HT content did not change the sympathoinhibitory effects of 8-OH-DPAT. These results suggested that 8-OH-DPAT has a direct effect on 5-HT1A receptors located in the VLPA in dogs.

Central and peripheral hypotensive effects of (+)- and (−)-nicardipine (1–10 μg/kg i.v. or i.c.v.) were investigated in normotensive and SH rats. The results suggest that a stereoselectivity exists for the peripheral and central hypotensive effects of the nicardipine isomers. (+)-Nicardipine was shown to be 3–8 times as potent as the (−) isomer in reducing blood pressure in all the experiments.

In anaesthetized, paralysed and artificially ventilated dogs, activities were recorded from the phrenic nerve and from respiratory units within the nucleus tractus solitarii (nTS), the nucleus ambiguus (nA) and the nucleus retroambigualis (nRA). The respiratory neurons were classified according to their discharge pattern and their response to lung inflation. Fentanyl injected into the vertebral artery (0.5–2 μg/kg) or intravenously (10 μg/kg) produced a depressant effect on the phrenic nerve motoneurons, on inspiratory cells (Iα and Iβ) and on phase-spanning expiratory-inspiratory neurons of the nTS and the nA. The duration of the inspiratory burst increased and the number of spikes and the peak activity were reduced. This pattern of inhibition was followed by complete blockade of spike genesis. Fentanyl also altered expiratory neurons: the duration of the expiratory discharge was enhanced. An increase followed by a decrease in the number of spikes per burst and a reduction in the peak acticity were observed. When the phrenic nerve was silent, continuous discharges appeared. High doses of fentanyl were needed to inhibit these tonic discharges. This pattern of inhibition concerns late peak expiratory units, expiratory units with a constant discharge pattern and the phase-spanning inspiratory-expiratory neurons. Naloxone antagonized these effects but induced the appearance of tonic discharges in fentanyl-treated phase-spanning expiratory-inspiratory neurons. Stimulation of peripheral chemoreceptors with almitrine (0.2 mg/kg i.v.) antagonized the effects of fentanyl. In addition, fentanyl facilitated the lung inflation reflex on respiratory neurons. In conclusion, fentanyl induces respiratory depression through a preferential effect on inspiratory neurons and alters the active inhibitory phase-switching mechanisms maintaining the expiratory periodicity. These experiments support the concept of an inhibitory influence of inspiratory neurons on expiratory cells and also support the idea that respiratory periodicity arises on a background of tonic activity for both inspiratory and expiratory neurons.

L-Glutamine acid and kainic acid injected int o the cisterna magna of dogs, produced a dose-dependent increase in blood pressure and a decrease in heart rate. In contrast, intravenous injection of both compounds was ineeffective. The hypertension was probably due to an increase in sympathetic tone as guanethidine prevented the rise in blood pressure induced by central administration of L-glutamic acid and kainic acid. Kainic acid was 1000 fold more potent than L-glutamic acid.

The haemodynamic effects of timolol and its inhibiting action on the cardiovascular and bronchial effects of isoproterenol have been studied. Splanchnic nerve activity was recorded. The antiarrhythmic action of timolol was studied on guinea pig isolated atria, using arrhythmias induced by epinephrine, ouabain or coronary ligation in the dog. Timolol is a very potent beta-adrenoceptor blocking agent, without specificity on beta1- or beta2-receptors. No intrinsic beta-stimulating or depressant effects were found. Timolol reduced splanchnic discharges. The antiarrhythmic effect of timolol was limited to epinephrine-induced arrhythmias.

The intravenous injection of loperamide induced an immediate fall in blood pressure and heart rate in anaesthetized rats. Both effects were inhibited by the opiate antagonists naloxone and MRZ 226 BS. Bilateral vagotomy also inhibited both effects whereas atropine only reduced the bradycardia, but the combination of atropine and tertatolol suppressed the bradycardia. A high dose of loperamide induced bradycardia in pithed rats. This effect was prevented by MRZ 2266 BS but not by naloxone. It is concluded that loperamide can elicit a vagally mediated reflex involving vagal and sympathetic mechanisms and could stimulate cardiac opiate receptors, probably κ, both effects leading to bradycardia.

Dextromoramide (0.01−0.2 mg/kg i.v.) and fentanyl (0.005−0.05 mg/kg) induced a dose dependent decrease in blood pressure and heart rate in dogs. These effects appear to be to a centrally mediated decrease in sympathetic tone, (i) Both drugs are 10−20 times more potent when injected into the vertebral artery or the cisterna magna than when they were injected intravanously, (ii) Both drugs reduced splanchnic nerve activity in intact animals. The effect appears to be localized to the medulla oblongata as suggested by central injections; transection of the brain stem at midpoint levels did not change the effects on blood pressure and heart rate induced by dextromoramide in intact bivagotomized dogs. After transection of the spinal cord at C1, dextromoramide induced a strong decrease in heart rate and a hypertension. Cutting of both vagus nerves abolished the decrease in heart rate and blood pressure. The hypotensive effect was therefore due to the bradycardia reducing cardiac output. In contrast in intact and midpontine dogs, a reduction in sympathetic tone contributes to the hypothensive effect. Nalorphine prevented and reversed the hypotensive, bradycardic and sympatho-inhibitory effects of dextromoramide. Piperoxan and yohimbine, two potent peripheral and central α-adrenoceptor blocking agents did not change the effects of dextromoramide. However oxotremorine or acetylcholine administered into the cisterna magna and vertebral artery administration of physostigmine abolished or reversed the haemodynamic effects of dextromoramide. A tropine abolished the antagonistic effects of cholinomimetic agents on the central cardiovascular effects of dextromoramide. Muscarinic mechanism appear to be involved in the haemodynamic effects of analgesic agents.

Opiate κ agonists were administered into the cisterna magna of normotensive urethane-anaesthetized artificially ventilated rats: ethylketocyclazocine (78.7 nM) (EKC) and dynorphin-(1–13) (62.3 nM) produced significant and long-lasting decreases in both blood pressure and heart rate. High doses of naloxone (1 mg/kg i.v.) were required to partially antagonize these effects. The central cardiovascular effects of EKC and dynorphin-(1–13) were compared to those of fentanyl (3 nM), [D-Ala 2 , Met 5 ]enkephalinamide (17 nM) and β-endorphin (2.9 nM) which induced increases in blood pressure and heart rate. These results suggest that opposite central cardiovascular effects could be induced by activation of various opiate receptors.

The effects of piribedil on responses to sympathetic stimulation were investigated in anaesthetized dogs. Piribedil (1 mg/kg i.v.) impaired the vasoconstrictor responses to lumbar sympathetic chain stimulation of the perfused hindlimb without changing the effects of noradrenaline. Piribedil (2 mg/kg i.v.) depressed the chronotropic responses to stimulation of the right anterior ansa and the inotropic response to stimulation of the left anterior ansa. Stimulation of the splanchnic nerve induced frequency dependent increases in systemic blood pressure. Piribedil antagonized this effect. Piribedil (1 mg/kg i.v.) attenuated the constrictor responses of the perfused mesenteric artery to postganglionic sympathetic stimulation and reduced the decreases in renal blood flow caused by stimulation of sympathetic renal nerves. The inhibitory efforts of piribedil were preferential on responses induced by low frequency stimulation of nerves. The hypertensive, vasoconstrictor and tachycardic effects of noradrenaline and tyramine were not affected. The effects of piribedil were reversed by haloperidol (0.5 mg/kg i.v.) or pimozide (0.2 mg/kg i.v.).

Dynorphin-(1–13) (i.v.) induced bradycardia and hypotension in artificially ventilated anaesthetized rats. These effects were prevented by MRZ 2266 BS. The bradycardia was inhibited by bilateral vagotomy whereas the fall in blood pressure was not sensitive to bilateral vagotomy and chlorpheniramine treatment. In pithed rats, dynorphin-(1–13) reduced heart rate. This bradycardia was prevented by MRZ 2266 BS but not by tertatolol and cimetidine. It is suggested that the dynorphin-(1–13)-induced effects result from the stimulation of central and cardiac κ-opiate receptors.

Xylazine (3–10 mg/kg), clonidine (1–3 mg/kg) and L.D.2855, three α-sympathomimetic agents with marked central effects, were found to be potent antinociceptive agents on the hot plate test in mice and on the test of electrical stimulation of rat's tail when administered intraperitoneally. In contrast, classical α-sympathomimetic agents, naphazoline, tetryzoline and oxymetazoline, were devoid of any antinociceptive action by parenteral route. Intracerebroventricular administration of xylazine, clonidine and L.D. 2855 induced an antinociceptive action at smaller doses than by intraparenteral route of administration. These results indicate a predominant central site of action. Intracerebroventricular administration of naphazoline in rats and of naphazoline, tetryzoline and oxymetazoline in mice, induced a strong antinociceptive effect. These results indicate that the failure to cross the blood-brain barrier was the cause of the ineffectiveness of classical α-sympathomimetic agents to cause analgesia. In conclusion, α-sympathomimetic mechanisms seem to induce antinociceptive action in rodents.

Intracerebroventricular injections of nicardipine (10 micrograms/kg) to SHR induced hypotension which was suppressed by pretreatment with 6-OHDA. This hypotension was also inhibited by a previous injection of naloxone (100 micrograms/kg i.c.v.) whereas the hypotension induced by intravenously injected nicardipine (10 micrograms/kg) was not changed. It is suggested that the release of endogenous opioids is involved in the sympatho-inhibitory effect of centrally administered dihydropyridine calcium channel antagonists.

The central cardiovascular effects of the calcium channel blocker nifedipine and the calcium channel activator BAY k 8644 were studied in anesthetized and ventilated normotensive Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHR). Both drugs were administered in a 1.5-microliter volume into the lateral ventricle of the brain (i.c.v.) or into the cisterna magna (i.c.). The injection of vehicle alone (i.c. or i.c.v.) did not significantly change mean arterial pressure (MAP) or heart rate. Nifedipine (5 and 50 micrograms/kg) and BAY k 8644 (5 and 50 micrograms/kg) induced opposite effects on MAP when centrally injected. Nifedipine decreased MAP and induced a bradycardia (i.c.v.) or no change in heart rate (i.c.), and BAY k 8644 increased MAP without any significant change in heart rate (i.c. or i.c.v.). These effects were more marked with the highest dose of either drug. These effects seemed to be of central origin, since they were suppressed by ganglionic blockade by hexamethonium (100 mg/kg i.v.), whereas after hexamethonium the hypotensive and the hypertensive responses to intravenously injected nifedipine and BAY k 8644, respectively, were preserved. Bilateral vagotomy suppressed the bradycardia induced by i.c.v. administered nifedipine. Previously i.c.v. administered nifedipine (5 micrograms/kg) antagonized the pressor response to BAY k 8644 (5 micrograms/kg i.c.v.). Changes in MAP and heart rate were significantly more marked in SHR than in WKY. These results indicate that a calcium channel inhibitor and a calcium channel activator can modulate in opposite fashion central mechanisms involved in blood pressure control.

Piperoxan (50-100 micrograms/kg) injected into the cisterna magna of anaesthetized dogs increased blood pressure, heart rate and resting rate of sympathetic nerve discharge. The baroreflex curve was shifted to the right with an elevation of the upper plateau (when blood pressure was lowered below resting values, renal sympathetic nerve activity rose to an upper plateau) and with no change in baroreflex sensitivity. Catecholamine depletion, produced by reserpine and alpha-methyl-p-tyrosine, did not change the effects of piperoxan on resting sympathetic nerve activity and on the baroreflex curve. Piperoxan (50 micrograms/kg i.c.) enhanced the rate of renal sympathetic nerve discharge in baroreceptor-denervated dogs. Piperoxan (5 micrograms) injected bilaterally into the nucleus tractus solitarii (NTS) increased blood pressure and resting sympathetic nerve activity with a shift of the sympathetic baroreflex curve to the right and no change in baroreflex sensitivity. Piperoxan (5 micrograms) injected bilaterally into the ventrolateral pressor area did not change blood pressure or the rate of sympathetic discharge. However, the baroreflex curve was shifted to the right with an elevation of the upper plateau level and the baroreflex sensitivity was increased. The present findings suggest that tonically active alpha 2-adrenergic mechanisms located within the NTS regulate resting and reflex sympathetic activity. An alpha 2-adrenergic mechanism in the rostral ventrolateral medulla modulates reflex sympathetic activity but has no influence on resting activity.

(Imidazolyl-2)-2-benzodioxane 1–4 (170 150) has been shown to possess α2-adrenoceptor blocking properties. The present investigation attempted to further characterize the α2-adrenoceptor blocking effect of 170 150. In pithed rats, 170 150 did not change the pressor effects of cirazoline a selective α1-adrenoceptor stimulant. On the other hand, 170 150 significantly and competitively antagonized the effects of B-HT 933, B-HT 920 and clonidine, showing that it has a potent action on α2-adrenoceptors since these three drugs act preferentially on α2-adrenoceptors.

SUMMARY 1. AR-C 239, a new α-adrenoceptor blocking drug, appears to act selectively on α1 sites in rats. At peripheral sites, this drug did not change the tachycardia induced by spinal sympathetic outflow stimulation in pithed rats, and did not antagonize the inhibitory effects of clonidine on this preparation. In addition, AR-C 239 showed predominant α1-adrenoceptor blocking properties in the bisected rat vas deferens preparation. AR-C 239 did not prevent or reverse the centrally mediated hypotensive and bradycardic actions induced by clonidine, in intact animals. 2. In conclusion, AR-C 239 seems to be a very useful tool for the characterization of peripheral and central α1-adrenoceptors, in this animal species.

Cloned neuroblastoma cell lines derived from the spontaneous mouse tumor C-1300 were used to study nerve cell differentiation. Our findings included a) morphologic and electrical differentiation was induced by the addition of dimethyl sulfoxide to the culture medium of some of the neuroblastoma clonal lines; b) a contrasting difference existed between the percentage of the phenylalanine-specific, tRNA species deficient in the peroxy Y-nucleoside in the mouse embryo or rat brain (6-10%) and that of mouse neuroblastoma cells (85%); c) the assembly of neuroblastoma microtubules and neurofilaments that are necessary for neurite outgrowth proceeded from preexisting pools of tubulin and actin, but a sustained level of phosphorylated tubulin was not required for this regulation; and d) the in vitro translation of tubulin and actin was accomplished with mRNA from rat brains in a wheat-germ cellfree system.

Intracerebroventricular (i.c.v.) injections of dihydropyridine derivatives calcium channel agonist (BAY K8644) and antagonist (nifedipine, nicardipine, PN 200-110) induced opposite long-lasting changes in blood pressure (BP) in pentobarbital anesthetized spontaneously hypertensive rats (SMR). I.c.v. nifedipine (NIF), nicardipine (NIC), and PN 200-110 decreased mean blood pressure dose-dependently and stereoselectively, (+) NIC and (+) PN being 8 and 3 times more potent than their (-) isomers, respectively. The decrease in BP was due to a withdrawal of the sympathetic tone, since NIF- and NIC-induced falls in BP were suppressed after either hexamethonium (HXM), 6 OHDA or bilateral adrenalectomy. I.c.v. BAY K8644 increased BP dose-dependently. The i.c.v. BAY K8644-induced hypertensive effect was inhibited: a), by NIF and (+) PN but not by (-) PN, therefore probably occurring at central DHP sites; b), by HXM and reserpine, thus probably mediated by an increase in sympathetic tone; c) by i.c.v. methylatropine (MA) while i.v. MA and i.c.v. HXM had no inhibitory effect, thus probably involving central muscarinic sites. In SHR, NIC did not after the K(+)-evoked ACh release but suppressed the BAY K8644-induced increase in ACh release. In anesthetized normotensive control rats (WKY), neither i.c.v. NIF, NIC or BAY increased BP and HR while, in conscious SHR it decreased BP without any change in HR. These data increased BP and HR while, in conscious SHR it decreased BP without any change in HR. These data suggest that central DHP sites may be involved in the cholinergic transmission and may participate in genetic hypertension via sympathetic tone.

Intracisternal (i. cist.) administration of d-tubocurarine (0.025-0. 100 mg. kg-1) in alpha-chloralose-anaesthetized dogs caused a dose-related increase in blood pressure associated with seizures. When injected in dogs pretreated with guanethidine (15 mg. i.v.), d-tubocurarine elicited the same effect while bilateral adrenalectomy abolished the pressor response. The hypotensive effect of clonidine (0.003 mg. kg-1 i. cist.) was reversed when clonidine was given after the alkaloid. These results suggest possbile interactions of the drugs at central GABA-ergic, glycinergic or cholinergic synapses.

Morphinomimetic peptides were injected into the cisterna magna of chloralosed dogs. Methionine enkepalin (500 microgram/kg i.c.) was found ineffective but beta-endorphin (50 microgram/kg i.c.) induced an initial and transient increase in blood pressure and heart rate followed by a delayed hypotension and bradycardia. The synthetic pentapeptides, [d-ala2]met-enkephalin (500 microgram/kg i.c.), [d-ala2]met-enkephalinamide 500 microgram/kg i.c.) also induced a marked hypotensive, bradycardic and sympatho-inhibitory effect. High doses of naloxone (100 microgram/kg i.v.) were required to antagonize these effects transiently.

In pentobarbital-treated dogs and rats, AR-C239, a new and potent alpha-adrenoceptor blocking drug, competitively antagonized pressor responses to adrenaline and inhibited pressor responses to noradrenaline, phenylephrine, tyramine, and dimethylphenylpiperazinium. Injected intravenously into closed-chest dogs, AR-C239 (3-50 micrograms/kg) induced a progressive fall in blood pressure, heart rate, and sympathetic nerve activity. The drug appears to be devoid of direct vasodilator action, and the fall in blood pressure results from the peripheral alpha-blockade. AR-C239 did not change the tachycardia induced by stimulation of the cardiac nerve in dogs and, at least in this preparation, seems to be a specific alpha 1-adrenoceptor blocking drug. When administered into the cisterna magna of dogs, AR-C239 did not have any centrally mediated cardiovascular actions and heart rate. AR-C239 did not modify the functioning of the baroreflex arc. Due to its specificity for alpha 1-Adrenoceptors, AR-C239 may be useful for characterizing alpha-adrenoceptors.

ET 495 and apomorphine, injected in small doses (0.1--1 microng/kg) into the femoral artery, induced a dose-dependent increase in femoral blood flow. This dilator effect was abolished by section of the ipsilateral femoral nerve and sciatic nerve, transection of the spinal cord, alpha-adrenoceptor blockade, ganglionic blockade and guanethidine. In addition, the increase in blood flow was inhibited by intravenous administration of haloperiol (2 mg/kg i.v.) or pimozide (2 mg/kg i.v.) and by injection of small doses (10--50 microng/kg) of these drugs into the femoral artery. It was concluded that a dopaminergic component located in the femoral vascular bed of the dog may be involved in the local vasodilator and sympathoinhibitory effect of apomorphine and ET 495.

The cytoplasmic and particulate tubulin content of postnatal rat brains was determined at various stages of development. The amount of tubulin in the soluble fraction was found to increase after birth and levels off at the age of 10–15 days, while the total protein content is still increasing. Indeed, the percentage of tubulin in the soluble fraction is about 33% at birth, stays at this value until day 10, and then decreases to 20% between days 10 and 15. On the other hand, the rate of increase in the level of the particulate tubulin parallels that of the total particulate proteins, and hence there is no change in the percentage of particulate tubulin during brain development. There was close agreement between the tubulin values obtained by the [ 3 H]-colchicine binding assay and those obtained by electrophoretic resolution in sodium dodecylsulfate-polyacrylamide gels. Polyacrylamide gel electrophoresis was also utilized to determine actin levels in developing brains. The percentage of cytoplasmic brain actin also decreased with the age of the rats, from a value of 20% at birth to 10% at day 30, while the percentage of the particulate actin remained constant. The decline in the percentage of cytoplasmic tubulin and actin during brain development can be accounted for by reduction in the proportions of the respective mRNA species. Translation of poly (A)-rich brain mRNA in a wheat-germ cell-free system showed that the percentages of tubulin and actin synthesized decreased gradually with age. Similar results were obtained by analyzing the proteins produced by isolated brain polysomes in a brain cell-free system.

In the vas deferens of the rat, (-)-170 150 (imidazolinyl-2)-benzodioxane-1-4 or 2-(2-(1,4-benzodioxanyl))-2-imidazoline appeared to be more selective than (+)-170 150 for antagonizing the inhibitory effects of clonidine on the twitch response. In contrast, (+)-170 150 was the most potent of the two isomers for antagonizing the centrally mediated clonidine- or azepexole-induced sleep in chickens. These unexpected results seem to indicate that there are several subpopulations of alpha 2-adrenoceptors.

Bromocolchicine, synthesized by substituting tho N-acetyl moiety of colchicine with a reactive bromoacetyl group, was found to be an affinity label for tubulin. Binding of [3H]colchicine to tubulin was competitively and irreversibly inhibited by bromocolchicine with a Ki value of 2.3 × 10−5 m . The affinity label could not be displaced by precipitating the protein with trichloroacetic acid and is thus covalently bound. Autoradiographs of brain high-speed supernatant proteins after their electrophoretic separation on sodium dodecyl sulphate/polyacrylamide gels showed that [3H]bromocolchicine reacted with four proteins, of which tubulin was one. Labelling of two of these proteins could be prevented by pretreatment of the brain extracts with α-bromoacetic acid, after which 70% of the covalently bound label was specifically located in the tubulin band. Up to 1.6 mol of affinity label could be bound per mol of tubulin, while under our experimental conditions 1 mol of protein bound irreversibly only 0.2 mol of [3H]colchicine. Autoradiography of sodium dodecyl sulphate/urea-polyacrylamide gels, which separate the subunits of tubulin, showed about 30% [3H] bromocolchicine bound to the α-subunit of tubulin and 70% to tho β-subunit. The irreversible binding site of colchicine was localized to the α-subunit, as labelling of only this subunit was inhibited by colchicine at high affinity label concentrations. At lower concentrations, colchicine inhibited the labelling of both subunits. Bromoacetic acid did not inhibit the reaction of the affinity label with the tubulin subunits, but increased the inhibition of [3H]bromocolchicine binding at lower concentrations of the affinity label in brain extracts preincubated with cold colchicine. This is interpreted to show a conformational change which takes place in the two subunits of tubulin upon binding of colchicine and results in the exposure of some of the binding sites of [3H]bromocolchicine to bromoacetic acid.

In anesthetized, normotensive beta-blocked rats, the alpha 1-adrenoceptor blocking drug, AR-C 239 (300 micrograms/kg, i.v.) induced a bradycardic effect related to a central increase in the vagal tone. This bradycardia was inhibited by previous administration of naloxone, intravenously (1 mg/kg) or centrally (100 micrograms/kg, i.c.) injected. Naloxone, by itself did not change the heart rate. In brainstem membranes from normotensive rats, AR-C 239 did not influence the stereoselective binding of [3H]naloxone. In spontaneously hypertensive (SH) rats, naloxone peripherally or centrally administered did not influence the activation of the vagal tone induced by AR-C 239, in beta-blocked animals. These results suggest the possible involvement of opiate release in the AR-C 239-induced vagal bradycardia, in normotensive rats. They also afford new arguments for the existence of close interactions between central alpha-adrenergic and opiate systems in the cardiovascular regulation. The possible participation of kappa-receptors in this effect is discussed. In addition, such an opiate mechanism triggered by central alpha 1-adrenoceptor blockade seems to be either absent or inactive in SH rats.

In cats anaesthetized with chloralose and urethane, administration of 1-dopa, 30–60 mg/kg, induced a decrease in blood pressure following an initial inconsistent and transient hypertension; splanchnic discharges were decreased. This inhibition of the sympathetic centres was abolished or reversed by piperoxane, 1–2 mg/kg, and yohimbine, 1 mg/kg, as was the sympatho-inhibitory effect of clonidine. In dogs, 1-dopa, 30 mg/kg, induced a marked hypertension followed by long lasting decrease. These results suggest a physiological role of the noradrenergic sympatho-inhibitory mechanism.

H. SCHMITT, H. SCHMITT and S. FENARD, Localization of the site of the central symphato-inhibitory action of l-DOPA m dogs and cats , European J. Phannacol. 22 (1973) 212–216. The hypotensive, bradycardic and sympatho-inhibitory effects of l-Dopa (20mg/kg, i.v.) injected after the peripheral Dopa decarboxylase inhibitor, M.K. 486 (20 mg/kg, i.V.), did not differ significantly in normal dogs and cats or in animals transected at midpontine level or at the upper border of the medulla oblongata. After peripheral Dopa decarboxylase inhibition by M.K. 486 (20 mg/kg, i.v.), there were no significant differences in the hypotensive effects of l-Dopa administered into the vertebral artery or intravenously in doses from 10 to 5 mg/kg. These results suggest that the main site of the sympatho-inhibitory effect of l-Dopa is in the medulla oblongata and/or the spinal cord.

Clonidine (150 Μg/kg s. c.) depressed avoidance conditioned reflexes in Long-Evans rats. Some α-adrenoceptor blocking agents piperoxan, tolazoline, phentolamine, dibenamine and phenoxybenzamine also depressed these reflexes. Yohimbine (1–2 mg/kg) was found to have no significant effect. Atropine (10 to 15 mg/kg i. p.) antagonized the depression of conditioned avoidance reflexes induced by α-adrenoceptor blocking agents as well as the depression produced by clonidine. Yohimbine (1–2 mg/kg) reduced the depressing effects of clonidine on avoidance conditioned reflexes. In rats treated with piperoxan (10 mg/kg) the effects of clonidine were reduced. In these rats the depression of conditioned reflexes was less than in rats treated with clonidine or piperoxan alone. These experiments suggest that clonidine depressed avoidance conditioned reflexes in rats by activating central α-adrenoceptors. It is proposed that α-adrenoceptor blocking agents might act as partial agonists.

Possible activation of α-adrenergic receptors at the level of the sympathetic centres in the brain stem by 2-(2,6-dichlorophenylamino)-2-imidazoline (St 155, Catapresan) was studied using α-adrenergic blocking drugs. The electrical activity of the preganglionic fibres of the splanchnic nerve was taken as an index of central sympathetic tone; St 155 was shown to produce a dose-dependent reduction of the splanchnic discharges. Phentolamine (1–5 mg/kg) and tolazoline (1–5 mg/kg) induced biphasic changes in splanchnic discharges; a strong inhibition concomitant with the fall in blood pressure was followed by a reduction. Small doses of phenoxybenzamine (1–5 mg/kg) increased splanchnic discharges, but high doses (10 mg/kg) reduced the discharges. An apparent reduction in the central inhibitory effect of St 155 was seen only when the splanchnic discharges were reduced by α-adrenergic blocking drugs. When the discharges had recovered, no antagonism could be found in spite of a strong α-adrenergic blockade. Intracerebro-ventricular injections of phentolamine did not antagonize the inhibitory effect of St 155 on vasomotor centres. The results and the possible presence of α-adrenergic receptors at the level of the sympathetic centres different from the peripheral ones are discussed.

The depressing action of a number of α-sympathomimetic agents on the central nervous system was studied in chickens and mice. Clonidine, 2-(2,6-dimethylphenylamino)-2-Δ2-oxazolidine 1–3, tetryzoline, naphazoline, tramazoline xylazine and oxymetazoline induced a loss of the righting reflex in chickens and prolonged the sleeping time induced by chloral in mice. These effects were dose-dependent and much more potent for the two former drugs. The actions in chickens and mouse were antagonized by previous treatment with four α-adrenergic blocking drugs (tolazoline, phentolamine, piperoxane, dibenamine) but not phenoxybenzamine. In chickens, chlorpromazine and imipramine also antagonized the action, but haloperidol was an antagonist only for naphazoline and tetryzoline. In mice, but not in chickens, atropine and mecamylamine were strong antagonists. These results suggest that activation of α-adrenergic receptors in the brain induces sedation and the involvement of cholinergic pathways in mice for this effect.

Clonidine (15 μg/kg, i.v.) induced an increase followed by a long-lasting decrease in blood pressure and reduced heart rate and cardiac output of dogs. All the indices of myocardial performance were decreased: maximal rate of rise in left ventricular pressure, max dP/dt/I.P. (I.P. = ventricular pressure at max dP/dt), pressure time index. The left ventricular end diastolic pressure was increased even when stroke work was decreased. The curve relating dP/dt to the developed pressure was flattened. These facts indicate that clonidine decreased myocardial contractility. Ventricular volumes were not altered. Pacing of the heart after clonidine did not lead to a recovery of cardiac output and myocardial contractility. To analyze the role of the sympathetic and vagal tones on these factors, 5 groups of dogs were used: (1) reserpinized, (2) reserpinized with both vagus nerves cut, (3) with β-adrenoceptor (S 2395, 50 μg/kg) and muscarinic receptor (atropine, 50 μg/kg) blockade, (4) with both vagus nerves cut, and (5)_with both carotid sinus nerves cut. Heart rate was markedly reduced in group 1 but not change in group 2 and 3 3; in groups 4 and 5, heart rate was decreased but to a smaller extent than in control dogs. Therefore the decrease in sympathetic tone and the increase in vagal tone were responsible for the bradycardia. The increase in the vagal tone was apparently due to potentiation of the influence of baroreceptor impulses. The maximal rate of rise in left ventricular pressure did not change in groups 1,2, and 3, but was decreased in groups 4 and 5, indicating that the loss of the sympathetic tone was responsible for the reduction in myocardial contractility. Cardiac output was not changed significantly in groups 2,3 and 4, but decreased in groups 1,3 and 5. The loss of the sympathetic tone and the bradycardia when very marked appear to reduce cardiac output. Left ventricular end diastolic pressure rose in groups 1 and 2, and was increased only transiently in groups 4 and 5. The decrease in myocardial contractility, the changes in blood pressure, the bradycardia and possibly the reduced venous return appear to be the factors influencing this parameter. Blood pressure shows the usual biphasic changes, an increase followed by a decrease in groups 3,4 and 5, did not change in group 1, and was only increased in group 2. The loss of the sympathetic tone was therefore responsible for this effect.

The effects of 2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride (ST 155) have been investigated on the peripheral sympathetic system. ST 155 induced an initial rise in blood pressure in normal and reserpinized or adrenalectomized animals, a contraction of the nictitating membrane of cats and isolated seminal vesicles of guinea pigs and a vasoconstriction in isolated ears of rabbits: all these actions were abolished by phentolamine, suggesting that ST 155 exerts these effects by direct stimulation of α-adrenergic receptors. No β-adrenergic blocking properties could be demonstrated. Low doses of ST 155 potentiated but higher doses decreased or even reversed the hypertensive actions of adrenaline and noradrenaline. The effects of the stimulation of the whole sympathetic system in pithed rats were reduced by ST 155, but the preganglionic stimulation of the nictitating membrane remained unaltered. These results are discussed and it is concluded that although ST 155 induced a certain degree of α-adrenergic blockade, this effect is not sufficient to account for the hypotensive action of the drug.

A 72 S ribosome can be isolated from EDTA-washed mitochondria of Saccharomyces cerevisiae. It easily dissociates into 38 S and 50 S subunits. Incorporation of amino acids into the protein of mitochondria purified in this way is shown to be inhibited by chloramphenicol, while cycloheximide has no effect. When EDTA treatment of the mitochondria is omitted, the mitochondrial fraction contains in addition a special class of 80 S ribosomes, presumably membrane-bound, which dissociate less easily than the “free” cytoplasmic ribosomes. Incorporation of amino acids into protein of these mitochondria is only partially inhibited by either chloramphenicol or cycloheximide and is stimulated by the addition of a cytoplasmic supernatant.

Clonidine and l-dopa (after M.K. 486) reduced pressor responses evoked by submaximal high frequency stimulation of the sciatic nerve, and medullary and hypotahalamic pressor areas. Low frequency stimulation at these sites and in the cervical spinal cord evoked responses in the splanchnic nerve consiting of early and late components which were inhibited by clonidine and l-dopa. Piperoxan or yohimbine reversed these effects. It is suggested that α-adrenoreceptor activation at medullary and spinal sites exerts sympatho-inhibitory effects.

Transection experiments and injection into the cerebral cavities were carried out in order to localize the site of action of St 155 in dogs and cats. Intercollicular decerebration and transection of the brain stem above the medulla oblongata did not change the bradycardia and hypertension followed by hypotension caused by St 155 nor was the reduction of the sympathetic discharges brought about by the drug affected. In contrast, transection of the spinal cord at C 1 level lowered blood pressure, abolished splanchnic discharges and suppressed the hypotensive effect of St 155. It is concluded that the medulla oblongata is the main site of the hypotensive and bradycardic actions of St 155. Low doses of St 155 injected into the cisterna magna or into the cerebral ventricle, lowered blood pressure, heart rate and reduced sympathetic discharges, but after a long latency period which precluded any conclusion about the localization of the site of action. It is suggested that this long latency period was due to the slow passage of the drug from the cerebro spinal fluid into the nervous tissue. Thus the central action of St 155 was confirmed and the medulla oblongata was found to be the main site of this action.

ET 495 is a new piperazine derivative inducing complex cardiovascular changes. In dogs infusion of low doses (20 μg/kg/min i.v.) or intraduodenal administration (2 mg/kg) induced a long lasting increase in femoral blood flow and decrease in splanchnic blood flow without changing blood pressure, heart rate, cardiac output and cerebral, coronary, or renal blood flow. By intravenous route (0.2 mg/kg), the peripheral vasodilatation followed a short hypertensive phase and polypnea. This hypertension appeared to be due to selective stimulation of cardioaortic chemoreceptors and was abolished by hexamethonium or by destruction of the chemoreceptors with acetic acid. This hypertension was accompanied by an increased discharge of sympathetic nerves; the discharge was more marked in the splanchnic nerve than in the lumbar sympathetic chain or in the cardiac nerve. Femoral vasodilatation was reversed by denervation of the lind limb, by ganglion blocking drugs and by depletion of catecholamines with reserpine or guanethidine. Moreover, the vasodilator phase was accompanied by a reduction of sympathetic discharges. This decreased sympathetic tome was more marked in the lumbar sympathetic chain than in the splanchnic nerve and less obvious in the cardiac nerve. The site of the reduced sympathetic tone was ascertained by localized injections. Injection of low doses of ET 495 into the main pulmonary artery induced hypotension, bradycardia and femoral vasodilatation. Injection of low doses into the femoral artery increased blood flow but after denervation of the hind limb, the same injection reduced the blood flow. Thus it appeared that ET 495 stimulated receptors located in the main pulmonary artery and femoral bed leading to reflex vasodilatation. In cat and rats ET 495, induced hypotension, bradycardia, reduction of respiration and decreased splanchnic discharges. These effects were reduced by bilateral vagotomy. It is concluded that ET 495 induces a selective increase in femoral blood flow in dogs by a shift from the splanchnic circulation.