Adrenergic receptor agonists induce the differentiation of pluripotent stem cell-derived hepatoblasts into hepatocyte-like cells.

Current induction methods of hepatocytes from human induced pluripotent stem cells (hiPSCs) are neither low cost nor stable. By screening a chemical library of 1,120 bioactive compounds and known drugs, we identified the α1-adrenergic receptor agonist methoxamine hydrochloride as a small molecule that promotes the differentiation of hiPSC-derived hepatoblasts into ALBUMIN+ hepatocyte-like cells. Other α1-adrenergic receptor agonists also induced the differentiation of hepatocyte-like cells, and an α1-receptor antagonist blocked the hepatic-inducing activity of methoxamine hydrochloride and that of the combination of hepatocyte growth factor (HGF) and Oncostatin M (OsM), two growth factors often used for the induction of hepatoblasts into hepatocyte-like cells. We also confirmed that treatment with methoxamine hydrochloride activates the signal transducer and activator of transcription 3 (STAT3) pathway downstream of IL-6 family cytokines including OsM. These findings allowed us to establish hepatic differentiation protocols for both mouse embryonic stem cells (mESCs) and hiPSCs using small molecules at the step from hepatoblasts into hepatocyte-like cells. The results of the present study suggest that α1-adrenergic agonists induce hepatocyte-like cells by working downstream of HGF and OsM to activate STAT3.

AV-block and conduction slowing prevail over TdP arrhythmias in the methoxamine-sensitized pro-arrhythmic rabbit model.

INTRODUCTION: The methoxamine-sensitized rabbit model is widely used to screen drugs for proarrhythmic properties, especially repolarization-dependent TdP arrhythmias. With the change of anesthesia and/or sensitizing agent, conduction disturbances have been reported as well. Therefore, we compared currently available in-house anesthetics in order to preserve arrhythmia sensitivity and preclude conduction disturbances. METHODS AND RESULTS: Rabbits were randomly assigned to 3 groups: (1) 35 mg/kg ketamine + 5 mg/kg xylazine; (2) 0.5 mL/kg hypnorm + 3 mg/kg midazolam; (3) 35 mg/kg ketamine + 20 mg/kg propofol. Anesthesia was maintained by 1.5% isoflurane. Concomitant infusion of methoxamine (17 µg/kg/min for 40 minutes) and dofetilide (10 µg/kg/min for 30 minutes) was used to induce arrhythmias. Sole methoxamine infusion exclusively decreased HR in groups 1 and 3. Dofetilide lengthened repolarization, followed in time by PQ/QRS prolongation, second-degree AV block, and subsequently TdP arrhythmias. TdP was seen in 80%, 0%, and 33% of the rabbits in groups 1, 2, and 3, respectively. Decreasing the dose of dofetilide to 5 µg/kg/min in ketamine/xylazine anesthetized rabbits resulted in a drop in TdP incidence (25%) while conduction disturbances persisted. Flunarizine (n = 6) suppressed all TdP arrhythmias while conduction disturbances remained present. CONCLUSION: TdP incidence in the methoxamine-sensitized rabbit could be dramatically influenced by anesthesia, drug dose, and flunarizine, while conduction slowing remained present. Thus, conduction slowing seems to be the integral outcome in this model.

Effects of Phikud Navakot extract on vascular reactivity in the isolated rat aorta.

The aim of the present study is to investigate the effect of standardized Phikud Navakot extract (NVKE) on aortic rings from male Sprague Dawley rats. Changes in tension were measured using an isometric force transducer and recorded on the PowerLab recording system. Vasorelaxant effect of NVKE was examined in the presence of indomethacin (10 microM), N(G)-nitro L-arginine methyl ester (L-NAME, 300 microM), methoxamine (0.1-300 microM), carbachol (1 nanoM-30 microM), or sodium nitroprusside (0.1 nanoM-10 microM). The results showed that NVKE (1-300 microg/mL) caused vasorelaxation in a concentration-dependent manner with a pEC50 value of 4.27 +/- 0.24 and R(max) of 67.7 +/- 13.9%. Pretreatment with indomethacin or L-NAME did not affect NVKE-induced vasorelaxation. However, co-incubation of indomethacin and L-NAME significantly reduced (p < 0.05) vasorelaxation to NVKE (100 microg/mL). Pre-treatment with NVKE significantly decreased (p < 0.05) endothelium-dependent relaxations to carbachol (R(max) = 52.90 +/- 14.3%), but not to sodium nitroprusside. Moreover contractions to methoxamine were unaffected after pretreatment with NVKE. The present study suggested that NVKE decreased vasorelaxation to carbachol in the rat aorta, which may exert at least against muscarinic receptors. These findings support the use of Phikud Navakot, a major ingredient of Yahom Navakot, against dizziness and fainting.

Impact of systemically active neurohumoral factors on the erectile response of the rat.

INTRODUCTION: Mean arterial pressure (MAP) and specific regulation of penile blood flow are the primary determinants of an erection. While this concept is well recognized, the differential relationship between systemically acting vasoactive factors on arterial pressure and erectile responses is not well described. AIM: The aim of this study was to determine how the modification of systemic levels of neurohumoral factors impacts on the magnitude and efficiency of the erectile response. MAIN OUTCOME MEASURES: The main outcome measures for this study are changes in MAP and intracavernosal pressure (ICP) following electrostimulation of the cavernous nerve. METHODS: Anesthetized adult, male Sprague-Dawley rats were catheterized for measuring MAP (carotid), ICP, and drug administration (vena cava). Erections were induced via cavernous nerve electrostimulation. Vasoactive drug infusions were used to produce changes in MAP levels including: hexamethonium, angiotensin II (ANGII)±hexamethonium, methoxamine±hexamethonium, losartan, MAHMA NONOate, and terbutaline. RESULTS: In general, ICP and MAP were linearly correlated regardless of treatment. Hexamethonium markedly dropped MAP and proportionately decreased the magnitude of the erectile response. ANGII or methoxamine given to hexamethonium-pretreated or untreated rats increased MAP similarly, but produced contrasting effects on erectile responses. ANGII-induced pressor responses were associated with increased erectile responses whereas all methoxamine treatments markedly decreased erectile responses. Depressor changes with losartan or terbutaline, but not MAHMA NONOate, also impacted negatively on the efficiency of the erectile responses at lower arterial pressures. CONCLUSIONS: In general, the magnitude of the erectile responses was found to be dependent upon the level of MAP, although the mechanism by which arterial pressure was changed impacted substantially on the characteristics of the relationship. The major finding was that circulation-wide α-adrenoceptor stimulation was extremely deleterious to erectile responses whereas global stimulation of ANG II receptors was actually proerectile. Overall, the results indicate that neurohumoral specificity in systemic hemodynamic control is also critical in establishing the optimal erectile environment in rats.

Beat-by-beat QT interval variability, but not QT prolongation per se, predicts drug-induced torsades de pointes in the anaesthetised methoxamine-sensitized rabbit.

INTRODUCTION: Accumulating evidence suggest that drug-induced QT prolongation per se poorly predicts repolarisation-related proarrhythmia liability. We examined whether beat-by-beat variability of the QT interval may be a complementary proarrhythmia marker to QT prolongation. METHODS: Anaesthetised rabbits sensitized towards developing torsades de pointes (TdP) were infused for 30 min maximum with explorative antiarrhythmic compounds characterised as mixed ion channel blockers. Based on the outcome in this model the compounds were classified as having a low (TdPlow; n=5), intermediate (TdPintermediate; n=7) or high (TdPhigh; n=10) proarrhythmic potential. Dofetilide (n=4) was included as a representative of a selective IKr-blocking antiarrhythmic with known high proarrhythmic potential. QT interval prolongation and beat-by-beat QT variability (quantified as the short-term variability, STV) were continuously assessed during the infusion or up to the point where ventricular proarrhythmias were induced. RESULTS: All compounds significantly prolonged the QT interval. For TdPlow and TdPhigh compounds the QT interval maximally increased from 169 ± 14 to 225 ± 28 ms (p<0.05) and from 186 ± 21 to 268 ± 42 ms (p<0.01), respectively. Likewise, in the dofetilide-infused rabbits the QT interval maximally increased from 177 ± 11 to 243 ± 25 ms (p<0.01). In contrast, whereas the STV in rabbits administered the TdPhigh compounds or dofetilide significantly increased prior to proarrhythmia induction (from 1.6 ± 0.4 to 10.5 ± 5.6 ms and from 1.6 ± 0.5 to 5.9 ± 1.8 ms, p<0.01) it remained unaltered in the TdPlow group (1.3 ± 0.6 to 2.2 ± 0.9 ms). In the TdPintermediate group, rabbits experiencing TdP had a similar maximal QT prolongation as the non-susceptible rabbits whereas the change in the STV was significantly different (from 0.9 ± 0.5 to 8.7 ± 7.3 ms vs 0.8 ± 0.3 to 2.5 ± 1.1 ms). DISCUSSION: It is concluded from the present series of experiments in a sensitive rabbit model of TdP that increased beat-by-beat QT interval variability precedes drug-induced TdP. In addition, assessment of this potential proarrhythmia marker may be useful in discriminating highly proarrhythmic compounds from compounds with a low proarrhythmic potential.

A combined cell membrane chromatography and online HPLC/MS method for screening compounds from Radix Caulophylli acting on the human alpha(1A)-adrenoceptor.

We have developed an online analytical method that combines alpha(1A)-adrenoceptor (alpha(1A)AR) cell membrane chromatography (alpha(1A)AR-CMC) with high performance liquid chromatography and mass spectrometry (HPLC/MS) for the identification of active components from Radix Caulophylli acting on the human alpha(1A)AR. Fractions retained by the alpha(1A)AR-CMC column were captured into a loop and the components were directly analyzed by combining an 8 port column switcher with an HPLC/MS system for separation and preliminary identification. Using methoxamine as a positive control drug, magnoflorine and caulophine from Radix Caulophylli were identified as the active molecules acting on the alpha(1A)AR. This new alpha(1A)AR-CMC-online-HPLC/MS method can be applied for screening active components acting on alpha(1A)AR from traditional Chinese medicines exemplified by Radix Caulophylli. This method will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.

Epinephrine, phenylephrine, and methoxamine induce infiltrative anesthesia via alpha1-adrenoceptors in rats.

AIM: To assess whether epinephrine, phenylephrine, and methoxamine act via certain subtypes of adrenoceptors to exert their local anesthetic activity. METHODS: We investigated cutaneous anesthesia from adrenoceptor agonists and/or antagonists in conscious, unanesthetized Sprague-Dawley male rats (weight 200-250 g). Cutaneous anesthesia was evidenced by a block of the cutaneous trunci muscle reflex, which is characterized by reflex movement of the skin over the back produced by twitches of lateral thoracispinal muscles in response to local dorsal cutaneous noxious pinprick. RESULTS: Local infiltration of epinephrine, L-phenylephrine, or methoxamine alone induces cutaneous anesthesia in rats in a dose-dependent way. Epinephrine is found to be 19 and 29 times more potent than those of methoxamine and L-phenylephrine, respectively. The cutaneous anesthesia induced by epinephrine, phenylephrine, or methoxamine can be significantly reduced by alpha(1)-adrenoceptor antagonists (eg, prazosin), alpha1, alpha2-adrenoceptor antagonist, alpha(1A)-adrenoceptor antagonist (eg, 5-methylurapdil), alpha(1B)-adrenoceptor antagonist (eg, chloroethylclonidine), or alpha(1D)-adrenoceptor antagonist (eg, BMY7873). CONCLUSION: Our results indicate that epinephrine, phenylephrine and methoxamine all act mainly via mixed subtypes of alpha(1)-adrenoceptors to induce cutaneous anesthesia in the rat.

Assessment of the ion channel-blocking profile of the novel combined ion channel blocker AZD1305 and its proarrhythmic potential versus dofetilide in the methoxamine-sensitized rabbit in vivo.

AZD1305 is a novel antiarrhythmic agent under clinical evaluation for management of atrial fibrillation. This study assessed its ion channel-blocking potency by the whole cell patch-clamp technique in vitro and its proarrhythmic liability in anesthetized methoxamine-sensitized rabbits in comparison with dofetilide. AZD1305 predominantly blocked the hERG, the L-type calcium and the hNav1.5 currents in a concentration-dependent manner. In vivo AZD1305 increased the QT interval (from 145 +/- 8 to 196 +/- 18 ms, P < 0.01) without inducing ventricular extrasystoles or torsades de pointes (TdP). In contrast, dofetilide prolonged the QT interval from 161 +/- 3 to 256 +/- 15 ms (P < 0.001) and caused TdP in 12/17 rabbits (P < 0.01 vs. AZD1305). During AZD1305 and dofetilide infusion, the QTend-peak interval maximally increased by 14 +/- 4 and 30 +/- 6 ms (P < 0.05 vs. AZD1305) and the beat-by-beat QT interval variability (quantified as the short-term variability, STV) changed from 2 +/- 0.8 to 2 +/- 0.3 ms (NS) and from 2 +/- 0.2 to 12 +/- 1.1 ms (P < 0.001), respectively. Following dofetilide-induced TdP, 6 rabbits each were injected with saline or AZD1305. In contrast to saline, AZD1305 abbreviated the QT interval (from 275 +/- 25 to 216 +/- 9 ms, P < 0.05), reduced the STV to 1 +/- 0.1 ms (P < 0.001) and suppressed TdP in all 6 rabbits (P < 0.01 vs. saline). In conclusion, AZD1305 can be characterised as a combined ion channel blocker that delays repolarization without increasing beat-by-beat variability of repolarization (BVR) or inducing TdP whereas selective IKr blockade by dofetilide prolongs the QT interval and eventually increases BVR resulting in TdP.

Noradrenergic control of GnRH release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study investigates the influence of alpha(1)-adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates alpha(1)-adrenoreceptor-GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media-alpha (MEM-alpha) at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to specific alpha(1)-adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1-10 mm). The alpha(1)-adrenoreceptor agonist (10 mm) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, alpha(1)-adrenoreceptor agonist (10 mm)-induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus-pituitary sequential double-chamber perifusion. Only after exposure of hypothalamic slices to alpha(1)-adrenoreceptor agonist (10 mm), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 +/- 12.3-36.2 +/- 21.6 ng/ml) confirming that the alpha(1)-adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.

Noradrenergic control of arginine vasopressin release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study aims at ascertaining the influence of alpha(1)-adrenoreceptors on arginine vasopressin (AVP) release in vitro and determine whether E(2) modulates the alpha(1)-adrenoreceptor and AVP interaction. Ten minutes after ewe killing, sagittal midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus with the median eminence, 2 mm thick, 2 per sheep) were dissected, placed in oxygenated minimum essential media-alpha (MEM-alpha) at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without E(2) (24 pg/ml). After 4 h equilibration, 10 min fractions were collected for 4 h interposed with 10 min exposure at 60 min to a specific alpha(1)-adrenoreceptor agonist or antagonist at various doses (0.1-10 mm). At the end of all perifusions, slices responded to KCl (100 mm) with AVP efflux (p < 0.05). Release of AVP was enhanced (p < 0.05) by the alpha(1)-adrenoreceptor agonist (methoxamine 10 mm; no E(2), n = 7 perifusion chambers: from 14.3 +/- 2.7 to 20.9 +/- 3.9, with E(2), n = 10: from 10.7 +/- 1.2 to 18.4 +/- 3.4 pg/ml) or the antagonist (thymoxamine 10 mm; no E(2), n = 5: from 9.5 +/- 3.1 to 30.4 +/- 6.0, with E(2), n = 10: from 10.8 +/- 0.9 to 39.1 +/- 6.3 pg/ml). With the agonist, the response occurred only at 80 min (p < 0.05) both in the presence and absence of E(2). Whereas, after the antagonist, values were higher (p < 0.05) throughout the post-treatment period (80-170 min) without E(2), but declined by 150 min in the presence of E(2). Furthermore, the response to the alpha(1)-adrenoreceptor antagonist was greater (p < 0.05; 90-140 min) than the agonist only in the presence of E(2). In conclusion, these results reveal direct alpha(1)-adrenoreceptor-mediated control of the hypothalamic AVP neuronal system which is modulated by E(2).

Disruption of prepulse inhibition after stimulation of central but not peripheral alpha-1 adrenergic receptors.

Prepulse inhibition (PPI) refers to the attenuation of startle when a weak prestimulus precedes the startling stimulus. PPI is deficient in several psychiatric illnesses involving poor sensorimotor gating. Previous studies indicate that alpha1 adrenergic receptors regulate PPI, yet the extent to which these effects are mediated by central vs peripheral receptors is unclear. The present studies compared the effects of intracerebroventricular (ICV) vs intraperitoneal (IP) delivery of several alpha1 receptor agonists on PPI. Male Sprague-Dawley rats received either cirazoline (0, 10, 25, 50 microg/5 microl), methoxamine (0, 30, 100 microg/5 microl), or phenylephrine (0, 3, 10, 30 microg/5 microl) ICV immediately before testing. Separate groups received either cirazoline (0, 0.25, 0.50, 0.75 mg/kg), methoxamine (0, 2, 5, 10 mg/kg), or phenylephrine (0, 0.1, 2.0 mg/kg) IP 5 min before testing. PPI, baseline startle responses, and piloerection, an index of autonomic arousal, were measured. Cirazoline disrupted PPI; effective ICV doses were approximately six times lower than effective IP doses. Methoxamine disrupted PPI after ICV infusion but failed to affect PPI with IP doses that were up to 30-fold higher than the effective ICV dose. Phenylephrine disrupted PPI with ICV administration, but did not alter PPI after IP injection of even a 20-fold higher dose. None of the ICV treatments altered baseline startle magnitude, but phenylephrine and methoxamine lowered startle after administration of high systemic doses. Piloerection was induced by cirazoline via either route of administration, and by IP methoxamine and phenylephrine, but not by ICV infusion of methoxamine or phenylephrine. These findings indicate that alpha1 receptor-mediated PPI disruption occurs exclusively through stimulation of central receptors and is dissociable from alterations in baseline startle or autonomic effects.

Fish Balistes capriscus skin extract-induced relaxation in mesenteric arterial bed of rat.

The vasorelaxing activity of the aqueous extract of fish Balistes capriscus skin (AEBc) on mesenteric arterial bed (MAB) of rats was studied. The bolus injections of AEBc (bolus of 5.1, 10.2, 20.5, and 41.1mg) significantly inhibited, in a concentration-dependent manner, the maximal contractile response induced by methoxamine (30 microM) in MAB. The vasodilatation action of AEBc is not mediated through beta-adrenoceptors or cyclo-oxigenase, since it was not affected by propranolol (20 microM) or diclofenac sodium (3 microM). The vasodilator response induced by subsequent addition of AEBc Balistes capriscus in bolus was significantly reduced in water infusion for endothelium removal. Treatment with an inhibitor of NO synthase (L-NAME, 10 microM) decreased AEBc effect. The guanylate cyclase inhibitor methylene blue (MB, 100 microM) had no significant effect on AEBc-induced vasodilatation. These results suggest that the vasorelaxing effect of AEBc is mediated by endothelium-dependent (NO/EDRF) and endothelium-independent neurally induced vasorelaxation from nonadrenergic and noncholinergic nerves (NO).

Molecular cloning and functional expression of the guinea pig alpha(1a)-adrenoceptor.

In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]prazosin and [(3)H]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [(3)H]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >>epinephrine>norepinephrine>methoxamine, and (b) for antagonists: prazosin> or 5-methyl-urapidil=benoxathian>phentolamine>>BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [(125)I-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [(3)H]inositol phosphates that was blocked by prazosin.

Systemic administration of calmodulin antagonist W-7 or protein kinase A inhibitor H-8 prevents torsade de pointes in rabbits.

BACKGROUND: The ventricular arrhythmia torsade de pointes (TdP) occurs after QT interval prolongation and is associated with sudden cardiac death. The afterdepolarizations that initiate TdP are facilitated by protein kinase A and the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase). METHODS AND RESULTS: In this study, we evaluated the feasibility of suppression of TdP through systemic therapy with kinase inhibitory agents in an established animal model. Under control conditions, TdP was inducible in 6 of 8 rabbits. CaM kinase blockade with the calmodulin antagonist W-7 reduced TdP in a dose-dependent fashion (4 of 7 inducible at 25 micromol/kg and 1 of 7 inducible at 50 micromol/kg). Increased intracellular Ca(2+) has been implicated in the genesis of afterdepolarizations, but pretreatment with high-dose W-7 did not prevent TdP in response to the L-type Ca(2+) channel agonist BAY K 8644 (300 nmol/kg), suggesting that CaM kinase-independent activation of L-type Ca(2+) current was not affected by W-7. Compared with control animals, W-7 reduced TdP inducibility without shortening the QT interval, increasing heart rate, or reducing the blood pressure. The protein kinase A antagonist H-8 also caused a dose-dependent reduction in TdP inducibility (5 of 6 at 1 micromol/kg, 4 of 6 at 5 micromol/kg, and 0 of 6 at 10 micromol/kg), but unlike W-7, H-8 did so by shortening the QT interval. CONCLUSIONS: These findings show that the acute systemic application of W-7 and H-8 is hemodynamically tolerated and indicate that kinase inhibition may be a viable antiarrhythmic strategy.

Alpha-adrenoceptor-mediated pressor responses in pithed rats fed diets with different calcium contents.

Results of many clinical and experimental studies indicate an inverse relationship between dietary calcium and the prevalence of hypertension. Our study was designed to evaluate the alterations in arterial blood pressure and the changes in alpha-adrenoceptor-mediated vascular reactivity in normotensive Sprague-Dawley and spontaneously hypertensive rats (SHR) fed from weaning (3 weeks of life) three diets: normal calcium (Ca 1%), low calcium (Ca 0.1%), and high calcium (Ca 2.5%). The systolic and the diastolic arterial blood pressures were measured weekly by the tail cuff method. The plasma calcium levels in the animals were also measured regularly by colourimetric methods, and the alpha-adrenoceptor-mediated vascular reactivity was evaluated by measuring the pressor responses to alpha-adrenoceptor agonists in pithed rats. These determinations were carried out at the end of the feeding periods (9 weeks of life in Sprague-Dawley rats and 20 weeks of life in SHR) and also at the moments when maximal differences in arterial blood pressure were observed between the conscious animals fed the normal calcium diet and those fed the other two diets. Dietary calcium deficiency increased arterial blood pressure in both strains but calcium supplements were effective to lower this only in hypertensive animals. The plasma calcium levels were altered in both strains when calcium administration was not normal. The low-calcium diet did not modify the pressor responses to either the alpha(1)-adrenoceptor agonist, methoxamine, or the alpha(2)-adrenoceptor agonist, B-HT 920 (5-allyl-2-amino-5,6,7, 8-tetrahydro-4H-thiazolo-(4,5-D)-acepin-dihydrochloride, talixepole), in the normotensive and the hypertensive rats. On the contrary, the high-calcium diet caused a definite decrease in alpha(1)- and alpha(2)-adrenoceptor-mediated vascular reactivity in both strains. The changes in the alpha-adrenoceptor-mediated vasoconstrictor responses were observed in pithed 9-week old Sprague-Dawley rats and in pithed 20-week old SHR, but none were observed in pithed 15-week old SHR, although at this age maximal differences in arterial blood pressure between the animals fed the high- and the normal calcium diet were observed. The results of this study suggest that the mechanisms implicated in the effects of dietary calcium supplements on arterial blood pressure are clearly different from the mechanisms, which bring about changes in arterial blood pressure when the diet is deficient in calcium. The results of this study also show that calcium administration causes variations in alpha-adrenoceptor-mediated vascular reactivity, but this is probably not the only mechanism implicated in the calcium effect on arterial blood pressure.

Supersensitivity with reduced capacity for pineal melatonin synthesis in constant light-treated rats.

Melatonin is synthetized from serotonin in two steps driven by the enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase. Constant light treatment reduces rat pineal hydroxyindole-O-methyltransferase activity while the activation of N-acetyltransferase becomes supersensitive to adrenergic stimulation. We studied the effect of this discrepancy on the production of melatonin. Male rats were kept under 12/ 12-h light/dark (LD) conditions or for 7 days under constant light (LL). They received subcutaneous injections of isoproterenol or methoxamine in the middle of the light period (LD-rats) or the estimated rest phase (LL-rats). A low dose of isoproterenol (0.1 mg/kg) increased pineal melatonin only marginally in LD-rats, while a maximum effect was found in LL-rats. A medium dose (0.2mg/kg) produced similar levels in both groups. A high dose (0.4 mg/kg) elevated pineal melatonin contents significantly more in normal than light-treated rats. Methoxamine (0.8 mg/kg) had no effects alone nor combined with isoproterenol. The results suggest supersensitivity with reduced capacity for melatonin formation in constant light-treated rats.

Adrenergic and cholinergic inputs in preoptic area of rats interact for sleep-wake thermoregulation.

Isolated studies have shown that both norepinephrine and acetylcholine into the medial preoptico-anterior hypothalamic area tonically regulate sleep-wake and body temperature. A possible interaction between these neurotransmitters for the regulation of such functions has been investigated in this study. To study this interaction a combination of either prazosin and carbachol or, scopolamine and methoxamine was injected into the medial preoptico-anterior hypothalamic area and the effect on sleep, wake, and rectal temperature recorded simultaneously. The combination of chemicals were selected based on our previous studies where it was observed that each of the chemicals in a combination had opposite effects. It was observed that injection of the combination expressed a resultant summated effects of individual component chemicals when injected in isolation (observed in previous studies). Because effect of neither of the chemicals in the combination was dominant, the results suggest an interaction and integration of the adrenergic and cholinergic inputs in the medial preoptico-anterior hypothalamic area for the regulation of sleep-wakefulness and body temperature.

Effects of methylenechloride-soluble fraction of Japanese angelica root extract, ligustilide and butylidenephthalide, on pentobarbital sleep in group-housed and socially isolated mice.

We previously showed that the extract of Japanese angelica root (JAR-E) reversed the decrease in pentobarbital (PB) sleep induced by isolation stress and yohimbine and methoxamine, stimulants of central noradrenergic systems, in mice. Here, we tested the effects of several fractions from JAR-E and ligustilide and butylidenephthalide, phthalide components of JAR-E, on PB sleep in isolated mice to elucidate the mechanism of the action of JAR-E. Methanol-soluble (Met-S) and -insoluble (Met-IS) fractions were obtained from JAR-E. Methylenechloride-soluble (MC-S) and -insoluble fractions (MC-IS) were prepared from Met-S. MC-S (11.4-76 mg/kg, p.o.) reversed the isolation stress-induced decrease in PB sleep, but neither Met-IS (0.8-2.4 g/kg, p.o.) nor MC-IS (0.7-2 g/kg, p.o.) had the same effect. The i.p. administration of MC-S exhibited a similar activity to that observed after the p.o. administration of the same fraction. Ligustilide (5-20 mg/kg, i.p.) and butylidenephthalide (10-30 mg/kg, i.p.) reversed PB sleep decrease in isolated mice. Both components (20 mg/kg, i.p.) attenuated the suppressive effects of yohimbine (30 nmol, i.c.v.), methoxamine (200 nmol, i.c.v.) and a benzodiazepine inverse agonist FG7142 (10 mg/kg, i.p.) on PB sleep in group-housed mice. These results suggest the contribution of ligustilide and butylidenephthalide to the effect of JAR-E on PB sleep in isolated mice, and implicate central noradrenergic and/or GABA(A) systems in the effects of these components.

Endothelium-dependent vasorelaxing activity of aqueous extracts of Ilex paraguariensis on mesenteric arterial bed of rats.

The effects of aqueous extracts of Ilex paraguariensis leaves (AEIp) were studied. Mesenteric arterial bed (MAB), precontracted by methoxamine with or without intact endothelium, was mounted on a tissue bath and exposed to plant extracts (bolus). The bolus injections of AEIp (300-1050 microg) significantly inhibited, in a concentration-dependent manner, the maximal contractile response induced by methoxamine (30 microm) in MAB. The endothelium-dependent relaxations were reversed by N(G)-nitro-L-arginine methyl ester (10 mM), whereas methylene blue (100 microM) was not capable of effectively inhibiting the AEIp-induced vasodilatation of MAB. The vasorelaxing effect of AEIp persisted in the presence of indomethacin (10 microM). These results suggest the involvement of NO of endothelial source (or others factors) in this vasodilatory effect.