Effects of agonists and phorbol esters on α1A-adrenergic receptor-Rab protein interactions.

In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.

Potential vascular α1-adrenoceptor blocking properties of metformin in rat aorta and tail artery.

Metformin is a widely used drug for the treatment of type 2 Diabetes Mellitus. Several studies have also suggested that metformin decreases blood pressure; although an interaction with α-adrenoceptors has been proposed, this mechanism needs to be further investigated. Since α1-adrenoceptors play a significant role to regulate vascular tone, this study has analysed the potential ability of metformin to block α1-adrenoceptors in rat aorta and tail artery. For this purpose, the contractile responses induced by noradrenaline, methoxamine, and phenylephrine were determined in the absence or presence of metformin in rat aorta and tail artery rings. In both arteries, noradrenaline, methoxamine, and phenylephrine produced concentration-dependent contractile responses. Interestingly, the contractile responses to noradrenaline, methoxamine, and phenylephrine were significantly and differentially blocked by metformin (1, 3.1 and/or 10 mM) but not by vehicle. These results suggest that metformin is capable to block α1-adrenoceptors and may explain, at least in part, the anti-hypertensive effect observed in several clinical trials.

Pharmacological evidence that metformin blocks the vasopressor responses mediated by stimulation of α1- and α2-adrenoceptors in pithed rats.

It has been reported that metformin reduces blood pressure although the mechanisms have not been described. Indeed, several mechanisms could be implicated including the interaction with α-adrenoceptors or inhibition of sympathetic outflow. Therefore, this study was designed to determine the capability of metformin to block the vasopressor responses induced by α1/2-adrenoceptor agonists or selective electrical stimulation of sympathetic outflow. For this purpose, Wistar male rats were anesthetized, pithed and cannulated for selective preganglionic stimulation of the vasopressor sympathetic outflow or drugs administration. The effect of i.v. bolus injection of metformin (180 and 310mg/kg) or its vehicle (bidistilled water) was studied on the vasopressor responses induced by: (1) selective sympathetic stimulation (0.03-3Hz); (2) exogenous noradrenaline (0.03-3µg/kg); (3) methoxamine (1-100µg/kg); and (4) UK 14,304 (0.1-30µg/kg). The tachycardic responses to noradrenaline were also investigated in presence of metformin. The vasopressor responses induced by selective electrical stimulation of sympathetic outflow were diminished by metformin (180 and 310mg/kg) and remained unchanged in presence of vehicle. Moreover, the vasopressor responses induced by exogenous noradrenaline, methoxamine and UK 14,304 were dose-dependently inhibited by i.v. bolus injections of metformin (180 and 310mg/kg) and were not affected by vehicle. Metformin practically did not block the tachycardic responses to noradrenaline except at the dose of 3µg/kg. Taken together, these results demonstrate that metformin is capable to block vascular α1/2-adrenoceptors but not cardiac ß-adrenoceptors. Thus, this mechanism could contribute, at least in part, on the hypotensive responses induced by metformin.

Evidence that chronic administration of 17ß-oestradiol decreases the vasopressor responses to adrenergic system stimulation in streptozotocin-diabetic female rats.

In vitro studies have indicated that 17ß-oestradiol exerts beneficial effects on the cardiovascular system by activating the nitric oxide pathway. However, these effects have not been demonstrated in vivo in the systemic vasculature of rats made diabetic through streptozotocin induction. Therefore, the goal of this study was to determine the effect of 17ß-oestradiol on vasopressor responses induced by sympathetic stimulation or i.v. injections of noradrenaline, methoxamine and B-HT 933 in sham-operated or ovariectomised, diabetic or non-diabetic female rats. Thus, rats were ovariectomised or sham-operated for this experiment. One week later, the animals were treated with streptozotocin (60mg/kg, i.p.) or its vehicle. Two weeks later, these rats were treated daily with 17ß-oestradiol (10µg/kg, s.c.) or its vehicle for five weeks. Next, under anaesthesia, the animals were pithed and prepared for blood pressure and heart rate measurements. 17ß-oestradiol failed to modify the vasopressor responses to (i) sympathetic stimulation, noradrenaline, methoxamine or B-HT 933 in sham-operated non-diabetic rats; (ii) sympathetic stimulation or B-HT 933 in sham-operated diabetic rats; (iii) noradrenaline or methoxamine in ovariectomised non-diabetic rats. In contrast, 17ß-oestradiol significantly decreased the vasopressor responses to (i) noradrenaline and methoxamine in sham-operated diabetic rats; (ii) sympathetic stimulation or B-HT 933 in ovariectomised non-diabetic rats; and (iii) sympathetic stimulation, noradrenaline, methoxamine or B-HT 933 in ovariectomised diabetic rats. These results suggest that chronic administration of 17ß-oestradiol decreases the vasopressor responses to adrenergic system stimulation in streptozotocin-induced diabetic rats. This report describes the first in vivo study reporting this effect of 17ß-oestradiol in diabetes.

Activation of 5-HT1B receptors inhibits the vasodepressor sensory CGRPergic outflow in pithed rats.

The importance of calcitonin gene-related peptide (CGRP) in the regulation of vascular tone has been widely documented. Indeed, stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses, which are mediated by CGRP release. These vasodepressor responses are inhibited by clonidine via prejunctional alpha(2A/2C)-adrenoceptors, but no study has yet reported the role of prejunctional 5-hydroxytryptamine (5-HT) receptors in this experimental model. Since activation of prejunctional 5-HT(1) receptors results in inhibition of neurotransmitter release, this study sets out to investigate as an initial approach the role of 5-HT(1B) receptors in the inhibition of the vasodepressor sensory outflow in pithed rats. Male Wistar pithed rats were pretreated with hexamethonium (2mg/kg.min) followed by i.v. continuous infusions of methoxamine (20 microg/kg min), and then by saline (0.02 ml/min) or CP-93,129 (a rodent 5-HT(1B) receptor agonist; 0.1, 1 and 10 microg/kg min). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T(9)-T(12)) resulted in frequency-dependent decreases in diastolic blood pressure. The infusions of CP-93,129, as compared to those of saline, inhibited the vasodepressor responses induced by electrical stimulation without affecting those to i.v. bolus injections of exogenous alpha-CGRP (0.1, 0.18, 0.31, 0.56 and 1 microg/kg). This inhibition by CP-93,129 was abolished by the antagonists GR127935 (5-HT(1B/1D)) or SB224289 (5-HT(1B)), but not by BRL15572 (5-HT(1D)). The above results suggest that CP-93,129-induced inhibition of the vasodepressor (perivascular) sensory outflow in pithed rats is mainly mediated by activation of prejunctional 5-HT(1B) receptors.

Evidence that some imidazoline derivatives inhibit peripherally the vasopressor sympathetic outflow in pithed rats.

Imidazoline derivatives (e.g. clonidine and moxonidine) and alpha(2)-adrenoceptor agonists (e.g. B-HT 933) have been shown to inhibit sympathetically-induced [(3)H]noradrenaline release in several isolated blood vessels. The present study has compared the potential capability of agonists at imidazoline I(1/2) receptors and/or alpha(1/2)-adrenoceptors to inhibit the sympathetically-induced vasopressor responses in pithed rats. For this purpose, male Wistar rats were pithed and prepared for measurement of diastolic blood pressure and heart rate. Then, the vasopressor responses induced by either selective electrical stimulation (2 ms, 60 V; 0.03, 0.1, 0.3, 1 and 3 Hz) of the vascular sympathetic outflow (T(7)-T(9)) or i.v. bolus injections of exogenous noradrenaline (0.03, 0.1, 0.3, 1 and 3 microg/kg) were determined before and during i.v. continuous infusions of the agonists B-HT 933 (alpha(2)), clonidine (alpha(2), I(1)), moxonidine (alpha(2), I(1)), cirazoline (alpha(1), I(2)), agmatine (putative endogenous ligand of imidazoline receptors) and methoxamine (alpha(1)), or equivalent volumes of physiological saline. Electrical sympathetic stimulation elicited frequency-dependent vasopressor responses which were significantly inhibited during the continuous infusions of B-HT 933, clonidine, moxonidine, cirazoline and agmatine, but not of physiological saline. Interestingly, the vasopressor responses to exogenous noradrenaline, which remained unaffected during the infusions of physiological saline, B-HT 933, moxonidine, cirazoline and agmatine, were significantly blocked during the infusions of clonidine or methoxamine. These results suggest that B-HT 933, moxonidine, cirazoline and agmatine induced a prejunctional inhibition of the vasopressor sympathetic outflow in pithed rats, whilst clonidine inhibited the vasopressor sympathetic outflow by both prejunctional and postjunctional mechanisms.

alpha1-Adrenoceptors in proximal segments of tail arteries from control and reserpinised rats.

It has been recently shown that the supersensitivity of distal segments of the rat tail artery to phenylephrine after chemical sympathectomy with reserpine results from the appearance of alpha(1D)-adrenoceptors. It is known that both alpha(1A)- and alpha(1D)-adrenoceptors are involved in the contractions of proximal portions of the rat tail artery. Therefore, this study investigated whether sympathectomy with reserpine would induce supersensitivity in proximal segments of the rat tail artery, a tissue in which alpha(1D)-adrenoceptors are already functional. Proximal segments of tail arteries from reserpinised rats were three- to sixfold more sensitive to phenylephrine and methoxamine than were arteries from control rats (n = 6-2; p < 0.05). The imidazolines N-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide (A-61603) and oxymetazoline, which activate selectively alpha(1A)-adrenoceptors, were equipotent in tail arteries from control and reserpinised rats (n = 4-2; p < 0.05), whereas buspirone, which activates selectively alpha(1D)-adrenoceptor, was approximately 4-fold more potent in tail arteries from reserpinised rats (n = 4-6; p < 0.05). Prazosin (nonselective) and 5-methylurapidil (alpha(1A)-selective), were competitive antagonists of contractions induced by phenylephrine and were equipotent in tail arteries from control and reserpinised rats (n = 4-6). The selective alpha(1D)-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) presented similar complex antagonism in tail arteries from control and reserpinised rats, with Schild slopes much lower than 1.0 (p < 0.05, n = 4-6). Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) revealed that mRNA encoding alpha(1A)-and alpha(1B)-adrenoceptors are similarly distributed in tail arteries from control and reserpinised rats, whereas mRNA for alpha(1D)-adrenoceptors is twice more abundant in the tail artery from reserpinised rats. In conclusion, the supersensitivity induced by reserpine is related only to alpha(1D)-adrenoceptors, even in tissues where this receptor subtype is already present and functional. Only the use of subtype-selective alpha(1)-adrenoceptor agonists detected the increased alpha(1D)-adrenoceptor component after reserpinisation, as the antagonists behaved similarly in tail arteries from control and reserpinised rats.

Antagonism by imidazoline-type drugs of muscarinic and other receptors in the guinea-pig ileum.

1 Several imidazolines were examined for the antagonism of muscarinic (M3) and other receptors on the isolated ileum of guinea-pig. The effect of the muscarinic agonist, carbachol was competitively antagonized by oxymetazoline at 10(-5) m. A dissociation constant (KB) of 3.6 microm for the antagonist was calculated. At higher concentrations, 3 x 10(-5) and 10(-4) m, of the antagonist, the agonist dose-response curve was shifted to the right with a decrease in the maximum effect. Thus, a non-competitive block occurred at higher concentrations of oxymetazoline. Blockade of histamine H, and serotonin receptor-mediated responses by oxymetazoline were also of a non-competitive type. 2 Naphazoline at 10(-4) m shifted the dose-response curves of carbachol and serotonin to the right by two- and 15-fold, respectively. The maximum contraction of the agonist was not affected. Tolazoline also had a weak antihistaminic activity. At similar concentration; tetrahydrozoline clonidine and phentolamine at 10(-5) m produced two-, three- and four-fold shift of the carbachol dose-response curve without significant changes in the maxima. Neither methoxamine, p-amino-clonidine nor cimetidine blocked the responses of carbachol. 3 The isosteric nature of the alpha-adrenoceptor agonist, oxymetazoline and some imidazolines with carbachol, in part, explains its molecular competition at the muscarinic M3 receptor of the guinea-pig ileum. Surprisingly, contractile effects of carbachol (M3), histamine (H1) or serotonin (5HT3/5HT4) were not influenced by methoxamine, tetrahydrozoline, p-amino clonidine and cimetidine.

Protein free diet feeding: effects on sympathetic activity and salivary evoked secretion in the submandibular gland of the rat.

Protein restriction impairs the salivary flow rate and composition in human and rats. The aim of the present work was to establish the effect of low protein (casein 5%) and protein free (casein 0%) isocaloric diets on sympathetic activity and salivary evoked secretion in the submandibular gland (SMG) of the rat. After 21 days, rats fed casein 0% presented: (a) a significant shift to the left of the dose-response curves (DRC) to the autonomic agonists-norepinephrine (NE), methoxamine, isoproterenol (ISO) and methacholine; (b) increased food consumption (p<0.001); (c) decreased body (p<0.001) and SMG (p<0.001) weights maintaining SMG/body (w/w) relation; (d) enhanced submandibular alpha1-adrenoceptor number without changes in the apparent dissociation constant (Kd); (e) increased submandibular NE content (p<0.05) and phosphoinositoside hydrolysis (p<0.001); (f) decreased submandibular tyrosine hydroxylase activity (TH) (p<0.01). Casein 5% feeding increased food consumption (p<0.01) and reduced body weight (p<0.05). This protein restriction increased metacholine-evoked salivation, but it altered neither submandibular sympathetic activity nor sympathetic-induced salivary secretion as compared to the Control group (C) fed a similar diet containing 25.5% protein. Present results suggest that in the adult rat, a protein free diet during 21 days lowers SMG sympathetic and cholinergic activity leading to supersensitivity as revealed by up-regulation of alpha1-adrenergic receptor number and increased autonomic-evoked salivation.

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).

Evidence for the role of alpha1D- and alpha1A-adrenoceptors in contraction of the rat mesenteric artery.

We investigated the alpha(1)-adrenoceptor subtype(s) involved in contraction of the isolated rat mesenteric artery by the use of the agonists noradrenaline (NA), phenylephrine (PHE), oxymetazoline (OXY), and methoxamine (MET), the competitive antagonists 8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)decane-7,9-dione dihydrochloride (BMY 7378) and 5-methylurapidil, and the alkylating agent chloroethylclonidine (CEC). Agonists showed the potency order NA> or =PHE>OXY>MET; pA(2) values for 5-methylurapidil and BMY 7378 were 7.74+/-0.11 and 8.72+/-0.28, respectively, while Schild slopes were not different than unity; alpha(1)-adrenoceptor alkylation with CEC showed a drastic decrease in maximal agonists-induced contraction and a shift to the right of about 46-, 122-, 2-, and 15-fold higher than controls for NA, PHE, OXY, and MET, respectively. Data suggest that alpha(1D)-adrenoceptors predominate for contraction in mesenteric artery of the Wistar rat, with a second population of alpha(1A)-adrenoceptors responding at high agonist concentrations.

Hormonal influence on expression and functionality of alpha1-adrenoceptor in rat submandibular gland.

In this work, we characterized alpha(1)-adrenoceptor expression and functionality in rat submandibular gland. Cumulative dose-response curve of methoxamine was constructed to determine the peroxidase secretion by glands from proestrous, estrous, metestrous and diestrous rats. They were compared to those from animals untreated or treated with sex hormones, estradiol and progesterone. The sensitivity of glands to an alpha(1)-adrenoceptor agonist varied depending on hormonal state, i.e. glands from proestrous and estrous were more sensitive to the stimulatory action of methoxamine than those from metestrous, diestrous and ovariectomised animals. The efficacy of the alpha(1) agonist was enhanced in glands from ovariectomised estrogen-treated rats but it was ineffective in glands from ovariectomised progesterone-treated rats. The functional studies correlated with 3H-prazosin binding assays in which estrogen increased alpha(1)-adrenoceptor density while progesterone decreased it. The results demonstrated that alpha(1)-adrenoceptor expression and functionality in rat submandibular glands are apparently under hormonal control and probably represent other examples of bidirectional interactions between neuronal and exocrine systems.

Cyclosporin A: effects on the secretory process and noradrenergic activity in the submandibular gland of the rat.

OBJECTIVES: The aim of the present work was to study the effect of long-term cyclosporine (CSA) administration on norepinephrine (NE) metabolism and adrenergic-evoked secretion in the rat submandibular gland (SMG). METHODS: Dose-response curves to adrenergic agonists (methoxamine, isoproterenol, NE) were performed in control and CSA (10 and 30 mg/kg every 2 days for 1 month)-treated rats after SMG duct cannulation. In SMG tissue neuronal NE uptake, release, synthesis and endogenous content were determined. In addition phosphoinositide intracellular signaling was also investigated. RESULTS: CSA administration caused an increase in salivary secretion evoked by methoxamine (alpha-adrenergic agonist) and NE but failed to modify salivation evoked by beta-adrenergic stimulation (isoproterenol). Long-term CSA administration decreased NE release and synthesis whereas it enhanced the amine uptake and phosphoinositide hydrolysis in the SMG. CONCLUSIONS: The administration of CSA for 30 days induced salivary gland sensitization likely mediated by diminished adrenergic input. Present results suggest that the decreased sympathetic activity evoked by long-term CSA administration in the rat SMG may lead to sensitization of the gland supported by increased phosphoinositide hydrolysis and enhanced adrenergic-evoked salivation.

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.

The importance of androgen on noradrenergic responses of vas deferens isolated from acutely stressed rats.

This study investigated the importance of androgen on responses to alpha and beta (norepinephrine) and alpha(1)(phenylephrine and methoxamine) agonists in vasa deferentia isolated from adult, immature, cryptorchid, and castrated rats submitted to swimming-induced acute stress. The participation of adrenergic nervous terminals was also investigated. Acute stress was shown to induce a significant subsensitivity to norepinephrine only in vas deferens from adult rats with normal levels of androgens. In addition, sympathetic denervation of the vas deferens prevented the appearance of subsensitivity. Subsensitivity was not seen when the experiments were carried out using phenylephrine and methoxamine. This shows that subsensitivity to norepinephrine in this acute stress situation may depend on other factors such as neuronal uptake, but not on alpha(1)-adrenoceptor response. Thus, when animals are exposed to acute stressogenic situations, this subsensitivity requires physiological levels of androgens to establish, and may also be involved in body homeostasis.

The androgenic effect of norethisterone and 5alpha-norethisterone on the contractile response of the rat vas deferens to methoxamine and serotonin.

Norethisterone (NET) and its metabolite 5alpha-norethisterone (5alpha-NET) are competitors for the androgen receptor. The sensitivity of the rat vas deferens to the contractile action of methoxamine and serotonin is regulated by hormonal and anatomical factors. The aim of this study was to evaluate the ability of NET and 5alpha-NET to induce the androgen-regulated contractile response to methoxamine and serotonin in the epididymal and prostatic portions of rat vas deferens. Adult male rats either intact, castrated or steroid-treated castrated were used. The contractility was recorded isometrically, and non-cumulative concentration-response curves to either methoxamine or serotonin were obtained. NET and 5alpha-NET partially restored the sensitivity to methoxamine and serotonin in the epididymal portion of castrated rats. The maximal responses to both agonists were significantly higher than those observed in castrated rats, and significantly lower than the responses observed in either intact or androgen-treated castrated rats. The prostatic portion was less responsive to both agonists than the epididymal portion, in all groups but castrated rats, as castration induced sensitivity to both agonists. NET and 5alpha-NET displayed a partial though similar androgenic activity in the rat vas deferens. These results contrast with previous reports where a decrease of androgenic effect due to the 5alpha-reduction of NET has been found.

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.

Adrenergic activation of phospholipase D in primary rat astrocytes.

Phospholipase D (PLD) activity was investigated in astrocytes prepared from newborn rat cerebral cortex using the transphosphatidylation assay. Basal PLD activity was measurable and was found to be enhanced by ATP, carbachol and noradrenaline. The activation by noradrenaline (EC50, 0.68 microM) was mimicked by methoxamine (EC50, 65 microM), an alpha 1-specific adrenergic agonist, and was inhibited by prazosine, an alpha 1-specific adrenergic antagonist. Clonidin, an alpha 2-adrenergic agonist, slightly lowered PLD activity whereas beta-adrenergic drugs were without effect. Experiments with mitogens indicate that PLD activation in astrocytes may be involved in the control of astrocytic cell proliferation.

Atrial natriuretic factor modifies the composition of induced-salivary secretion in the rat.

We have previously reported that although the atrial natriuretic factor (ANF) was not a sialogogic agonist, it enhanced cholinergic, alpha-adrenergic and peptidergic (substance P) stimulated salivation in the submaxillary and parotid gland of the rat. The purpose of the present work was to study whether ANF modified the composition of agonist-induced saliva in the rat. Results showed that in the submaxillary gland, ANF increased sodium and decreased potassium excretion when salivation was stimulated by methacholine (MC) or substance P (SP). However, when salivation was induced by methoxamine (MX), ANF only increased sodium excretion. On the other hand, in the parotid gland, ANF increased both sodium and potassium excretion when salivation was induced either by MC or SP but did not modify electrolyte output in MX induced salivary secretion. Protein output and amylase activity were not modified by the presence of ANF when the aforementioned sialogogic agonists were used to elicit salivation in either gland. Although ANF did not modify the volume of isoproterenol (IP) induced saliva, it increased protein output in both glands and it increased amylase activity in the parotid gland. The present results suggest that ANF may play a role in the modulation of salivary secretion in the parotid and submaxillary glands of the rat. ANF effect is likely to be mediated by modifications in the calcium level linked to phosphoinositide metabolism within the acinar and/or the ductal cells of the salivary glands.

Alpha 1-adrenoceptors mediating contraction in arteries of normotensive and spontaneously hypertensive rats are of the alpha 1D or alpha 1A subtypes.

Alpha 1-Adrenoceptor subtypes mediating contraction in carotid, aorta, mesenteric and caudal arteries from both Wistar Kyoto (WKY) normotensive and spontaneously hypertensive (SHR) rats were investigated by using the alpha 1A-adrenoceptor agonist methoxamine and antagonized with selective, competitive antagonists WB-4101, 5-methyl urapidil or BMY 7378 (8-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4,5)decane -7,9-dione dihydrochloride). Isometric tension changes were recorded after methoxamine addition to the arterial rings, and the effects of the antagonists determined. All the antagonists shifted to the right the concentration-response curve to methoxamine. pA2 values indicate that all arteries but caudal express the alpha 1D-adrenoceptor subtype, since BMY 7378 values were high in these arteries. Due to the high pA2 values for 5-methyl urapidil and WB-4101 and the low values for BMY 7378 we conclude that the tail artery expresses the alpha 1A and not the alpha 1B subtype. No differences were found between both strains of rats, suggesting that hypertension does not modify the alpha 1-adrenoceptors in conductance arteries.