Stimulation Of Alpha-adrenergic Receptors Research Articles

Norepinephrine-induced stimulation of p38 mitogen-activated protein kinase is mediated by arachidonic acid metabolites generated by activation of cytosolic phospholipase A(2) in vascular smooth muscle cells.

p38 mitogen-activated protein kinase (MAPK) is activated by norepinephrine (NE) in the vasculature and is implicated in vascular smooth muscle hypertrophy, contraction, and cell migration. NE promotes influx of Ca(2+) and activates cytosolic phospholipase A(2) (cPLA(2)) in vascular smooth muscle cells (VSMC). The purpose of this study was to determine the contribution of cPLA(2)-generated arachidonic acid (AA) and its metabolites to the activation of p38 MAPK measured by its phosphorylation, in response to NE in rabbit VSMC. NE-induced p38 MAPK activation was found to be mediated through the stimulation of alpha-1 and alpha-2 adrenergic receptors, was dependent on extracellular Ca(2+), and was attenuated by an inhibitor of cPLA(2) (pyrrolidine-1). Moreover, the cPLA(2) product, AA, activated p38 MAPK in VSMC. p38 MAPK activation elicited by NE was decreased significantly by the lipoxygenase (LO) inhibitor baicalein, and to a lesser extent by the cytochrome P450 inhibitor 17-octadecynoic acid, but was not affected by the cyclooxygenase inhibitor indomethacin. The LO metabolites of AA, namely 5(S)-hydroxyeicosatetraenoic acid (HETE), 12(S)-HETE, and 15(S)-HETE and the cytochrome P450 metabolite 20-HETE, activated p38 MAPK. NE-induced p38 MAPK stimulation was found to be independent of phospholipase D (PLD) activation in rabbit VSMC. Transactivation of the epidermal growth factor receptor (EGFR) by NE also did not contribute to p38 MAPK activation. These data suggest that cPLA(2)-generated AA and its LO metabolites mediate NE-induced p38 MAPK stimulation in rabbit VSMC by a mechanism that is independent of PLD and EGFR activation.

Cardiovascular regulation during hypoxia in embryos of the domestic chicken Gallus gallus.

Renewed interest in the use of the embryonic chicken as a model of perinatal cardiovascular regulation has inspired new questions about the control mechanisms that respond to acute perturbations, such as hypoxia. The objectives of this study were to determine the cardiovascular responses, the regulatory mechanisms involved in those cardiovascular responses, and whether those mechanisms involved the central nervous system (CNS) of embryonic chickens. Heart rate (f(H)) and blood pressure were measured in chicken embryos of different incubation ages during exposure to different levels of hypoxia (15, 10, and 5% O(2)). At all levels of hypoxia and at all developmental ages, a depression of f(H) and arterial pressure was observed, with the exception of day 20 embryos in 15 and 10% O(2). The intensity of the embryonic f(H) and blood pressure responses were directly related to the level of hypoxia used. Muscarinic and alpha-adrenergic receptor stimulation limited the hypoxic hypotension on days 15-19 and 15-21, respectively, as indicated after blockade with atropine and phentolamine. During the final 3 days of incubation, the intensity of the hypoxic hypotension was magnified due to alpha-vasodilation caused by beta-adrenergic and muscarinic receptor stimulation. In 19- to 21-day-old embryos, the f(H) response to hypoxia was limited by alpha-adrenergic receptor stimulation as indicated by the accentuated bradycardia after blockade with phentolamine. Furthermore, on day 21, atropine limited the hypoxic bradycardia, indicating that muscarinic receptors also play a role in the f(H) response at this age. In addition, the muscarinic actions on the heart and the adrenergic effects on the vasculature appeared to occur through a hypoxic-induced direct release from chromaffin tissue and autonomic nerve terminals. Thus, in embryonic chickens, the only cardiovascular response to hypoxia that involves the CNS was the cholinergic regulation of arterial pressure after day 15 of incubation. Therefore, although embryonic chickens and fetal sheep, the standard models of perinatal cardiovascular physiology, respond to hypoxia with a similar redistribution of cardiac output, the underlying mechanisms differ between these species.

Cell signaling and ion transport across the fish gill epithelium

A large array of circulating and local signaling agents modulate transport of ions across the gill epithelium of fishes by either affecting transport directly or by altering the size and distribution of transporting cells in the epithelium. In some cases, these transport effects are in addition to cardiovascular effects of the same agents, which may affect the perfusion pathways in the gill vasculature and, in turn, affect epithelial transport indirectly. Prolactin is generally considered to function in freshwater, because it is the only agent that allows survival of some hypophysectomized fish species in freshwater. It appears to function by either reducing branchial permeability, Na,K-activated ATPase activity, or reducing the density of chloride cells. Cortisol was initially considered to produce virtually opposite effects (e.g., stimulation of Na,K-activated ATPase and of chloride cell size and density), but more recent studies have found that this steroid stimulates ionic uptake in freshwater fishes, as well as the activity of H-ATPase, an enzyme thought to be central to ionic uptake. Thus, cortisol may function in both high and low salinities. Growth hormone and insulin-like growth factor appear to act synergistically to affect ion regulation in seawater fishes, stimulating both Na,K-activated ATPase and Na-K-2Cl co-transporter activity, and chloride cell size, independent of their effects on growth. Some of the effects of the GH-IGF axis may be via stimulation of the number of cortisol receptors. Thyroid hormones appear to affect seawater ion regulation indirectly, by stimulating the GH-IGF axis. Natriuretic peptides were initially thought to stimulate gill ionic extrusion, but recent studies have not corroborated this finding, so it appears that the major mode of action of these peptides may be reduction of salt loading by inhibition of oral ingestion and intestinal ionic uptake. Receptors for both arginine vasotocin and angiotensin have been described in the gill epithelium, but their respective roles and importance in fish ion regulation remains unknown. The gill epithelium may be affected by both circulating and local adrenergic agents, and a variety of studies have demonstrated that stimulation of alpha-adrenergic versus beta-adrenergic receptors produces inhibition or stimulation of active salt extrusion, respectively. Local effectors, such as prostaglandins, nitric oxide, and endothelin, may affect active salt extrusion as well as gill perfusion. Recent studies have suggested that the endothelin inhibition of salt extrusion is actually mediated by the release of both NO and prostaglandins. It is hoped that modern molecular techniques, combined with physiological measurements, will allow the dissection of the relative roles in ion transport across the fish gill epithelium of this surprisingly large array of putative signaling agents.

Critical role of alpha1-adrenergic receptors in acute and sensitized locomotor effects of D-amphetamine, cocaine, and GBR 12783: influence of preexposure conditions and pharmacological characteristics.

Psychostimulant-induced locomotor hyperactivity is commonly associated with an inhibition of dopamine reuptake. However, a physiological coupling between noradrenergic and dopaminergic neurons occurring through the stimulation of alpha1-adrenergic receptors has recently been proposed. This possibility was tested on locomotor responses induced either by D-amphetamine and cocaine, which both interfere with noradrenergic and dopaminergic transmissions, or by GBR 12783, a specific dopamine reuptake inhibitor. In an attempt to control the effects of stress and novelty on noradrenergic neurons activity, rats were submitted to habituation procedures consisting of either a 15-h period of habituation to the experimental environment ("long-habituation") or to repeated exposure to intraperitoneal saline injections for 3 consecutive days ("three-session"). Three-session-exposed animals exhibited a pronounced locomotor reactivity to saline injection which did not occur after noradrenergic depletion, clonidine (20 microg/kg) or prazosin (0.5 mg/kg) pretreatments, or in long-habituation-preexposed animals. Cocaine and GBR 12783 locomotor hyperactivities were doubled in three-session vs. long-habituation-preexposed rats, whereas D-amphetamine responses were similar in both conditions. Prazosin (0.5 mg/kg) pretreatment reduced the acute locomotor effects of the three psychostimulants in both procedures and blocked the behavioral sensitization induced by repeated injections of D-amphetamine (0.75 mg/kg) or cocaine (5 mg/kg). GBR 12783 (5 mg/kg) failed to induce significant behavioral sensitization. In addition to their role in the acute and sensitized locomotor responses to psychostimulants possessing different pharmacological characteristics, alpha1-adrenergic receptors are involved in animal reactivity to previously experimented procedures. This suggests an implication of noradrenergic neurons in the vulnerability to psychostimulants.

Reactive Oxygen Species Mediate Alpha-adrenergic Receptor-stimulated Hypertrophy in Adult Rat Ventricular Myocytes

Norepinephrine (NE) causes hypertrophic growth of cardiac myocytes via stimulation of alpha1-adrenergic receptors (α1-AR). Reactive oxygen species (ROS) can act as signaling molecules for cell growth. Accordingly, we tested the hypothesis that ROS mediate α1-AR-stimulated hypertrophic growth in adult rat ventricular myocytes (ARVM). NE increased the level of intracellular ROS as assessed by lucigenin chemiluminescence or cytochrome c reduction, and this effect was prevented by the superoxide dismutase (SOD)-mimetic MnTMPyP. NE also caused the induction of MnSOD mRNA. α1-AR stimulation with NE (1 μM) in the presence of propranolol (2 μM) for 48–96 h caused a hypertrophic growth phenotype characterized by a 36±3% increase in3H-leucine incorporation, a 49±14% increase in protein accumulation, a six-fold induction of atrial natriuretic peptide mRNA, actin filament reorganization, and the induction of MnSOD mRNA. These responses were all prevented by pretreatment with the α1-AR-selective antagonist prazosin (100 nM ) or the SOD-mimetics MnTMPyP (50 μM) and Euk-8 (100 μM). MnTMPyP had no effect on α1-AR-stimulated3H-inositol phosphate turnover or the hypertrophic phenotype caused by the protein kinase C activator phorbol-12-myristate-13-acetate. Thus, ROS play a critical role in mediating the hypertrophic growth response to α1-AR-stimulation in ARVM.

Viral croup.

Viral croup.

Effect of alpha1-adrenergic stimulation of Cl- secretion and signal transduction in exocrine glands (Rana esculenta).

In the present work, the effect of stimulation of alpha-adrenergic receptors on Cl- secretion via exocrine frog skin glands was investigated. The alpha-adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the beta-adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl- secretion was measured as the amiloride-insensitive short circuit current (ISC). Addition of noradrenaline induced a biphasic increase in the ISC. The increase in ISC coincided with an increase in the net 36Cl- secretion. The noradrenaline-induced increase in ISC was dose-dependent with an EC50 of 13 +/- 0.3 microM. Epifluorescence microscopic measurements of isolated, fura-2-loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca2+]i) response. Application of noradrenaline induced a biphasic [Ca2+]i response, which was dose-dependent with an EC50 of 11 +/- 6 microM. The Ca2+ plateau unlike the peak-response was sensitive to removal of Ca2+ from the extracellular medium. The noradrenaline-induced increase in the Cl- secretion as well as in [Ca2+]i was sensitive to the alpha1-adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca2+]i indicating that the release was independent of ryanodine-sensitive Ca2+ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5-trisphosphate (IP3) content suggesting that the signal transduction pathway leading to the noradrenaline-induced increase in Ca2+ involved IP3 and a release of Ca2+ from IP3-sensitive stores.

Intrathecal dexmedetomidine attenuates hypercapnic but not hypoxic cerebral vasodilation in anesthetized rabbits.

Systemic dexmedetomidine (DXM) attenuates the cerebral vasodilation induced by hypercapnia and decreases the cerebral blood flow response to hypoxia. We determined whether lumbar intrathecal DXM affected the cerebrovascular reactivity to hypercapnia and hypoxia. Rabbits (n = 55) anesthetized with pentobarbital were prepared for measurement of pial vessel diameters using a closed cranial window preparation. The first study evaluated the response to hypercapnia after intrathecal administration of DXM (2 microg/kg; n = 7) or normal saline (n = 8). The second evaluated the response to hypercapnia after intrathecal DXM in the presence of yohimbine (20 microg/kg followed by DXM 2 microg/kg; n = 7). The third evaluated the response to mild or moderate hypoxia after intrathecal DXM (2 microg/kg; n = 7) or normal saline (n = 7). The hypercapnic responses were also examined in the presence of systemic DXM (2, 10 microg/kg; n = 6), topical DXM (10-8 m, 10-6 m; n = 6) and of intrathecal clonidine (2 microg/kg; n = 7). The pial arteriolar dilator response to hypercapnia was significantly attenuated after intrathecal administration of DXM. Pretreatment with yohimbine completely blocked the decreased reactivity to hypercapnia. Intrathecal clonidine, although less than DXM, also attenuate the hypercapnic response. Intrathecal DXM did not affect the vasodilation of pial arterioles induced by mild or moderate hypoxia. The systemic DXM 10 microg/kg and topical DXM 10-6 m, but not systemic 2 microg/kg and topical 10-8 m, attenuated hypercapnic vasodilation of pial arterioles. The presence of alpha2-adrenoceptor agonist administered intrathecally into the lumbar spinal region attenuates hypercapnic but not hypoxic cerebral vasodilation, probably via a stimulation of central alpha2-adrenergic receptors of the central nervous system.

Platelet-derived growth factor inhibits alpha1D-adrenergic receptor expression in vascular smooth muscle cells in vitro and ex vivo.

Indirect evidence suggests that stimulation of alpha1-adrenergic receptors (ARs) increases smooth muscle cell (SMC) growth in the growing and adult artery and worsens atherosclerosis and restenosis after balloon injury. In support of a direct adrenergic effect, we have previously shown that alpha1D-AR stimulation induces SMC hypertrophy in cell and vessel organ culture. Because interactions between alpha1-ARs and peptide growth factors may be important in normal and pathological SMC growth, herein we examined regulation of alpha1D-AR expression by growth factors. Platelet-derived growth factor (PDGF)-BB dose- and time-dependently lowered alpha1D mRNA in cultured quiescent SMCs (e.g., 58% inhibition at 20 ng/ml, 24 h, p <.05), whereas other alpha1-AR transcripts were unaffected. This same selective effect was seen in the medial layer of aorta in ex vivo organ culture. However, PDGF-AA, insulin-like growth factor-1, insulin, epidermal growth factor, endothelin, histamine, and serotonin had no effect, whereas thrombin induced a modest (1.8-fold) increase. PDGF-BB inhibition of alpha1D-AR mRNA was accompanied by a 42% reduction in total alpha1-AR density (p <.05) and a functional decrease in norepinephrine-mediated protein synthesis. alpha1D mRNA half-life was not significantly affected by PDGF-BB (3.8 versus 3.2 h). However, transcriptional activity of the alpha1D promoter was inhibited. Reduction in alpha1D-AR mRNA depended partly on new protein synthesis, and was abolished by protein kinase C inhibition, whereas phosphatidylinositol 3 kinase and mitogen-activated protein kinase kinase inhibition had no effect. These data demonstrate that PDGF-beta receptor stimulation (because PDGF-AA had no effect) induces a selective inhibition of alpha1D-AR expression and hence norepinephrine-mediated SMC growth. This down-regulation may lessen additive or synergistic growth effects of catecholamines with other growth factors in vascular hypertrophic diseases.

α1-Adrenergic Hypothesis for Pulmonary Hypertension

Pulmonary hypertension (PH) is a chronic and disabling condition that affects the pulmonary vasculature. Once PH is diagnosed, the prognosis is generally poor with a rapid downhill course. PH management is largely empirical because the underlying pathophysiologic mechanisms that are responsible for the excessive vasoconstrictor and vascular smooth muscle proliferative responses are poorly understood. Based on new information concerning the role of adrenergic receptors in regulating various cellular functions, a new perspective on the genesis of PH has emerged, along with a unifying hypothesis for the role of alpha1-adrenergic receptors present in the pulmonary vasculature as the major contributor to the pathophysiologic changes associated with PH. Adrenergic receptors that are present on vascular smooth muscle cells regulate vascular tone and growth. The alpha1-adrenergic receptors that are present on the small- and medium-sized pulmonary arteries have a unique and greatly enhanced affinity and activity to alpha1-adrenergic agonists. Under physiologic conditions, this helps in regulating vascular tone and maintains an adequate ventilation/perfusion matching. However, the excessive stimulation of alpha1-adrenergic receptors produces not only smooth muscle contraction but also proliferation and growth. The conditions that produce an increase in alpha1-adrenoreceptor gene synthesis, density, and activity (such as hypoxia or changes in vessel wall pressure) or increase the levels of its agonists (such as norepinephrine, appetite suppressants, or cocaine) greatly enhance pulmonary vascular smooth muscle contractile and proliferative responses and lead to the development of PH. An understanding of the role played by these receptors in the pathophysiology of PH would not only help to avoid the use of alpha1-agonists for appetite suppression and other disease states, but also would help in developing new drugs to block these receptors. A further understanding of the alpha1-adrenoreceptor subtypes present in the pulmonary vasculature, the factors that regulate their expression, and their intracellular signaling pathways would help researchers to devise newer therapeutic strategies and, hopefully, to find a cure for this crippling condition.

Adrenergic and vasopressinergic contributions to the cardiovascular response to acute hypoxaemia in the llama fetus.

1. The effects of fetal intravenous treatment with phentolamine or a vasopressinergic V1-receptor antagonist on the fetal cardiovascular responses to acute hypoxaemia in the llama were investigated. 2. Six llama fetuses were surgically prepared between 60 and 70 % of gestation under general halothane anaesthesia with vascular catheters and transit-time ultrasonic flow probes around a carotid artery and a femoral artery. At least 4 days after surgery all fetuses were subjected to a 3 h experiment: 1 h of normoxia, 1 h of hypoxaemia and 1 h of recovery while on slow i.v. infusion with saline. On separate days this experiment was repeated with fetal i.v. treatment with either phentolamine or a V1-receptor antagonist dissolved in saline. 3. During saline infusion all llama fetuses responded to acute hypoxaemia with intense femoral vasoconstriction. Phentolamine during normoxia produced hypotension, tachycardia and vasodilatation in both the carotid and the femoral circulations. During hypoxaemia, fetuses treated with phentolamine did not elicit the pronounced femoral vasoconstriction and all died within 20 min of the onset of hypoxaemia. A V1-receptor antagonist produced a femoral vasodilatation during normoxia but did not affect the fetal cardiovascular responses to acute hypoxaemia. 4. In conclusion, alpha-adrenergic and V1-vasopressinergic mechanisms contribute to a basal vasoconstrictor tone in the femoral circulation in the llama fetus. The enhanced femoral vasoconstriction during acute hypoxaemia in the llama fetus is not mediated by stimulation of V1-vasopressin receptors, but is dependent on alpha-adrenergic receptor stimulation. Such alpha-adrenergic efferent mechanisms are indispensable to fetal survival during hypoxaemia in the llama since their abolition leads to cardiovascular collapse and death.

Alpha1-adrenergic, D1, and D2 receptors interactions in the prefrontal cortex: implications for the modality of action of different types of neuroleptics.

The activation of rat mesocortical dopaminergic (DA) neurons evoked by the electrical stimulation of the ventral tegmental area (VTA) induces a marked inhibition of the spontaneous activity of prefrontocortical cells. In the present study, it was first shown that systemic administration of either clozapine (a mixed antagonist of D1, D2, and alpha1-adrenergic receptors) (3-5 mg/kg, i.v.), prazosin (an alpha1-adrenergic antagonist) (0.2 mg/kg, i.v.), or sulpiride (a D2 antagonist) (30 mg/kg, i.v.), but not SCH 23390 (a D1 antagonist) (0.2 mg/kg, i.v.), reversed this cortical inhibition. Second, it was found that following the systemic administration of prazosin, the VTA-induced cortical inhibition reappeared when either SCH 23390 or sulpiride was applied by iontophoresis into the prefrontal cortex. Third, it was seen that, whereas haloperidol (0.2 mg/kg, i.v.), a D2 antagonist which also blocks alpha1-adrenergic receptors, failed to reverse the VTA-induced inhibition, the systemic administration of haloperidol plus SCH 23390 (0.2 mg/kg, i.v.) blocked this inhibition. Finally, it was verified that the cortical inhibitions obtained following treatments with either "prazosin plus sulpiride" or "prazosin plus SCH 23390" were blocked by a superimposed administration of either SCH 23390 or sulpiride, respectively. These data indicate that complex interactions between cortical D2, D1, and alpha1-adrenergic receptors are involved in the regulation of the activity of prefrontocortical cells innervated by the VTA neurons. They confirm that the physiological stimulation of cortical alpha1-adrenergic receptors hampers the functional activity of cortical D1 receptors and suggest that the stimulations of cortical D1 and D2 receptors exert mutual inhibition on each other's transmission.

Functional cross-talk between endothelial muscarinic and alpha2-adrenergic receptors in rabbit cerebral arteries.

Interactions between two classes of receptors have been observed in several cell lines and preparations. The aim of this work was to assess the impact of simultaneous stimulation of endothelial muscarinic and alpha2-adrenergic receptors (alpha2-AR) on vascular reactivity. Rabbit middle cerebral arteries were isolated and changes in isometric tension were recorded in the presence of indomethacin. Inhibition of nitric oxide (NO) synthase with Nomega-nitro-L-arginine (L-NOARG, 100 micromol l(-1)) revealed alpha-AR-dependent contractions. Pre-addition of acetylcholine (ACH, 1 micromol l(-1)) augmented oxymetazoline (OXY, 10 micromol l(-1), alpha2-AR agonist)-, but decreased phenylephrine (PE, 10 micromol(-1), alpha1-AR agonist)-induced contraction (P<0.05). The effects of ACH were endothelium-dependent. Vessels were precontracted with 40 mmol l(-1) KCl-physiological salt solution (PSS) in the absence of L-NOARG, or PE or OXY in the presence of L-NOARG. In the presence of high external K+ or PE, ACH induced a potent relaxation (P<0.05). In the presence of OXY, however, ACH mediated contraction (P<0.05). After pertussis toxin (PTX, inactivator of Galpha(i/o) proteins) pre-treatment, alpha2-AR-dependent contractions were abolished. Forty mmol l(-1) KCl-PSS induced contraction was not altered by PTX whereas ACH-induced relaxation was augmented (P<0.05). To investigate if endothelin-1 (ET-1) intervened in the endothelium-dependent contractile response to ACH in the presence of OXY-dependent tone, vessels were incubated in the presence of BQ123 (1 micromol l(-1)), an ETA receptor antagonist. OXY-mediated tone was not affected by BQ123; however, ACH-induced contraction was reversed to a relaxation (P<0.05). These data indicate that activation of endothelial alpha2-AR triggers an endothelium-dependent, ET-1 mediated, contraction to ACH. This suggests that activation of alpha2-AR affects muscarinic receptor/G protein coupling leading to an opposite biological effect.

Effects of alpha1-adrenergic stimulation on L-type Ca2+ current in rat ventricular myocytes.

The effect of alpha1-adrenergic stimulation on L-type Ca2+ current (ICa,L) in adult rat ventricular myocytes was investigated using three different methods of current recording. During conventional whole-cell recordings with 5 mm-BAPTA included in the pipette solution, phenylephrine (20 microM) did not increase ICa,L after 10 min of application. With nystatin perforated-patch whole-cell recordings, phenylephrine potentiated ICa,L, although there were variations among myocytes. The most frequent response was a transient suppression of peak ICa,L at approximately 2 min of exposure followed by a sustained increase of current amplitude evident after 5-10 min exposure. The relative current amplitude 10 min after phenylephrine application was 1.08+/-0.05 compared to control (n=14 cells,P<0.05). During cell-attached single channel recordings, phenylephrine (1 microM) increased the L-type Ca2+ channel open probability (NPo) by 2.25+/-0.31-fold (n=21,P<0.01). It potentiated NPo by increasing the number of openings per sweep and also by promoting longer openings. These effects developed slowly in approximately 10 min. Phenylephrine had no on unitary current amplitude. The potentiation was also elicited by methoxamine (5 microM) and was blocked by prazosin (1 microM), indicating that it was mediated by alpha1-adrenergic receptor stimulation. The increase in NP(o) was suppressed by chelerythrine, a protein kinase C inhibitor. Our results demonstrate that ICa,L can be enhanced by alpha1-adrenergic stimulation, and stress the importance of not disturbing the intracellular environment during studies of the modulation of cardiac ICa,L by alpha1-adrenergic stimulation.

Dopamine regulates phosphate uptake by opossum kidney cells through multiple counter-regulatory receptors.

The purpose of this study was to determine the mechanisms of dopamine regulation of phosphate uptake in opossum kidney (OK) cells, a model of proximal renal tubules. Dopamine stimulated cAMP generation and inhibited radiolabeled phosphate uptake into OK cell monolayers by 14.4 +/- 1.8%. The effect of dopamine was transient, as phosphate uptake returned toward control level by 3 h despite the continued presence of dopamine. Pretreatment with pertussis toxin increased dopamine inhibition of phosphate uptake to 25 +/- 3%, increased the duration of the dopamine effect to at least 3 h, and enhanced cAMP generation. In an OK cell clone that overexpressed cAMP phosphodiesterase, dopamine did not inhibit phosphate uptake, but pharmacologic inhibition of protein kinase A activation did not prevent dopamine inhibition of phosphate uptake. A DA1 receptor agonist inhibited phosphate uptake more potently than dopamine (29.5 +/- 1.1%) or a DA2 receptor agonist (7.9 +/- 2%). However, both DA1 and DA2 receptor antagonists completely blocked dopamine inhibition of phosphate uptake. DA1, but not the DA2, antagonists blocked dopamine-stimulated cAMP generation. Treatment with alpha-adrenergic receptor antagonists potentiated dopamine inhibition of phosphate uptake to the same extent as pertussis toxin and was not additive with pertussis toxin. It is concluded that dopamine inhibits phosphate uptake through DA1 and DA2 receptor stimulation by cAMP-dependent and -independent pathways and activates a pertussis toxin-sensitive counter-regulatory pathway that attenuates this response through alpha-adrenergic receptor stimulation.

Gβγ-independent Coupling of α2-Adrenergic Receptor to p21rhoA in Preadipocytes

In preadipocytes, alpha2-adrenergic receptor (alpha2-AR) stimulation leads to a Gi/Go-dependent rearrangement of actin cytoskeleton. This is characterized by a rapid cell spreading, the formation of actin stress fibers, and the increase in tyrosyl phosphorylation of the focal adhesion kinase (pp125(FAK)). These cellular events being tightly controlled by the small GTPase p21(rhoA), the existence of a Gi/Go-dependent coupling of alpha2-AR to p21(rhoA) in preadipocytes was proposed. In alpha2AF2 preadipocytes (a cell clone derived from the 3T3F442A preadipose cell line and which stably expresses the human alpha2C10-adrenergic receptor) alpha2-adrenergic-dependent induction of cell spreading, formation of actin stress fibers, and increase in tyrosyl phosphorylation of pp125(FAK) were abolished by pretreatment of the preadipocytes with the C3 exoenzyme, a toxin which impairs p21(rhoA) activity by ADP-ribosylation. Conversely, C3 exoenzyme had no effect on the alpha2-adrenergic-dependent increase in tyrosyl phosphorylation and shift of ERK2 mitogen-activated protein kinase. alpha2-Adrenergic stimulation also led to an increase in GDP/GTP exchange on p21(rhoA), as well as to an increase in the amount of p21(rhoA) in the particulate fraction of alpha2AF2 preadipocytes. Stable transfection of alpha2AF2 preadipocytes with the COOH-terminal domain of betaARK1 (betaARK-CT) (a blocker of Gbeta gamma-action), strongly inhibited the alpha2-adrenergic-dependent increase in tyrosyl phos- phorylation and shift of ERK2, without modification of the tyrosyl phosphorylation of pp125(FAK) and spreading of preadipocytes. These results show that alpha2-adrenergic-dependent reorganization of actin cytoskeleton requires the activation of p21(rhoA) in preadipocytes. Conversely to the activation of the p21(ras)/mitogen-activated protein kinase pathway, the alpha2-adrenergic activation of p21(rhoA)-dependent pathways are independent of the beta gamma-subunits of heterotrimeric G proteins.

Stimulation of alpha-1 adrenergic receptors facilitates spatial learning in rats

The present experiments were designed to examine the effects of alpha-1 adrenergic stimulation and inhibition on memory encoding and to investigate whether the alpha-1 adrenergic and muscarinic cholinergic systems interact in the regulation of spatial navigation behavior in the Morris water maze test and we also studied the effects of d-cycloserine, a partial agonist at the glycine binding site on the N-methyl- d-aspartate (NMDA) receptor complex, on the performance of scopolamine-treated rats in this task. Pre-training subcutaneous administration of St-587 (a putative alpha-1 agonist) at 1000 μg kg −1 or 1500 μg kg −1 improved water maze navigation to a hidden platform. Prazosin (an alpha-1 antagonist), 300–2000 μg kg −1, did not significantly impair the spatial navigation performance. Pre-training administration of prazosin 1000 μg kg −1, but not 300 μg kg −1, slightly potentiated the deficit in water maze navigation seen after scopolamine (200 μg kg −1, pre-training intraperitoneal injection). Pre-training administration of St-587 at a dose 1500 μg kg −1, but not 500 μg kg −1, slightly ameliorated the scopolamine-induced (200 μg kg −1) impairment in performance of rats. Pre-training administration of prazosin at doses 300 or 1000 μg kg −1 or St-587 at doses 500 μg kg −1 or 1500 μg kg −1 did not have any significant influence on the scopolamine-induced (200 μg kg −1) increase of swimming speed. Furthermore, d-cycloserine at the dose of 300 μg kg −1 but not 1000 or 3000 μg kg −1 reversed the scopolamine (200 μg kg −1)-induced deficit in acquisition of the water maze task but not the increase in motor output (increased swimming speed). These results indicate that the stimulation of alpha-1 adrenoceptors may facilitate the encoding of new information. These findings suggest that alpha-1 adrenergic mechanisms do not participate or at least are not the most critical part of the noradrenergic system in the interaction between noradrenaline and muscarinic receptors in the modulation of learning and memory. In addition, these results suggest that d-cycloserine may be effective in alleviating states of central cholinergic hypofunction.

Effect of phenylephrine, endothelin and angiotensin II on reperfusion arrhythmias. A role for Na+/H+ exchanger activation via protein kinase C

Stimulation of receptors for alpha 1-adrenergic agonist, endothelin (ET) and angiotensin II (AT) activates the cardiac sarcolemmal Na+/H+ exchanger (NHE), perhaps via protein kinase C(PKC)-mediated pathway(s). We tested for the ability of these extracellular stimuli to exacerbate reperfusion arrhythmias and for the possible role of NHE activation and PKC in such phenomena. Isolated rat hearts (n = 12/group) were subjected to dual coronary perfusion. After 15 min of aerobic perfusion, flow to the left coronary bed was reduced to 5% of basal values for 12 min, and the same bed was then reperfused for 5 min. An alpha 1-adrenergic agonist phenylephrine (PE) at 1 or 10 mumol/L, ET at 0.5 or 5nmol/L or AT at 1 or 10mumol/L was infused selectively into the left coronary bed during 12 min of regional low flow ischemia. The incidence of reperfusion-induced ventricular fibrillation (VF) was increased from 17% in control to 33% and 75%* with 1 and 10 mumol/L PE(*p < 0.05 vs control) from 8% in control to 8% and 12% with 0.5 and 5 nmol/L of ET. However, AT had no effect. The selective NHE inhibitor NOE642 at 1 mumol/L, infused concomitantly with 10 mumol/L PE, reversed the proarrhythmic effects of PE; VF incidence was reduced from 67% to 8%*. However, glibenclamide (a blocker for the ATP-sensitive K+ channel) at 1 mumol/L did not affect the proarrhythmic effects of PE. Infusion of a specific PKC inhibitor GF109203X(GF) at 30 or 300 nmol/L, starting from 5 min before ischemia and maintained throughout ischemia concomitantly with 10 mumol/L of PE, was partially effective in reducing VF incidence; which reduced from 75% in control to 42% with 300 nmol/L of GF. These results suggest that, in rat hearts subjected to regional low-flow ischemia and reperfusion, stimulation of alpha 1-adrenergic receptor can exacerbate reperfusion-induced VF, whose mechanism(s) may involve NHE activation. Moreover, PKC activation does not appear to be the sole signaling mechanism for this phenomenon.

Sensitization of the cardiac Na channel to alpha1-adrenergic stimulation by inhalation anesthetics: evidence for distinct modulatory pathways.

Alpha1-adrenergic receptor stimulation has been shown to inhibit cardiac Na+ current (INa). Furthermore, some form of synergistic interaction of alpha1-adrenergic effects on INa in combination with volatile anesthetics has been reported. In this study, the authors investigated the possible role of G proteins and protein kinase C in the effects of halothane and isoflurane in the absence and presence of alpha1-adrenergic stimulation on the cardiac INa. The standard whole-cell configuration of the patch-clamp technique was used. INa was elicited by depolarizing test pulses from a holding potential of -80 mV in reduced Na+ solution (10 mM). The experiments were conducted on ventricular myocytes enzymatically isolated from adult guinea pig hearts. The inhibitory effect of halothane (1.2 mM) and isoflurane (1 mM) on peak INa was significantly diminished in the presence of guanosine 5'-O-[2-thiodiphosphate (GDPbetaS). In myocytes pretreated with pertussis toxin (PTX), the potency of halothane was significantly enhanced, but the isoflurane effect was unchanged. In the presence of the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIS), the effect of halothane was unchanged. In contrast, the effect of isoflurane on INa in the presence of BIS was significantly enhanced. The positive interaction between methoxamine and halothane was evident in the presence of G protein and PKC inhibitors. In contrast, the effect of methoxamine with isoflurane was additive in the presence of GDPbetaS or BIS. Different second messenger systems are involved in the regulation of cardiac Na+ current by volatile anesthetics. The effect of halothane involves a complex interaction with G proteins but is independent of regulation by PKC. In contrast, PKC is involved in the modulation of cardiac INa by isoflurane. In addition, non-PTX-sensitive G proteins may contribute to the effects of isoflurane. The positive interaction between methoxamine and anesthetics are independent of G proteins and PKC for halothane. In the case of isoflurane, the positive interaction with methoxamine is coupled to PTX-insensitive G proteins and PKC.

Dopamine

Dopamine is a biogenic amine synthesized in the hypothalamus, in the arcuate nucleus, the caudad, and various areas of the central and peripheral nervous system. It has been widely established that dopamine and its agonists play an important role in cardiovascular, renal, hormonal, and central nervous system regulation through stimulation of alpha and beta adrenergic and dopaminergic receptors. There are several agonists of dopamine-2 (DA 2 ) dopaminergic receptors, such as bromocriptine, pergolide, lisuride, quinpirole, and carmoxirole, which inhibit norepinephrine release and produce a decrease in arterial blood pressure; in some cases, bromocriptine and pergolide also reduce heart rate. From a therapeutic point of view, the above-mentioned agonists are used for treating Parkinson's disease, acting over DA 2 dopaminergic receptors of the nigrostriatal system. Bromocriptine and the other dopaminergic agonists mentioned act over DA 2 receptors of the tuberoinfundibular system, inhibiting prolactin release and decreasing hyperprolactinemia and tumor size. Among DA 1 receptor agonists, we can mention fenoldopam, piribedil, ibopamine, SKF 3893, and apomorphine (nonspecific). Activation of these receptors decreases peripheral resistance, inducing lowering of arterial blood pressure and increases in heart rate, sympathetic tone, and activity of the renin aldosterone system. Among DA 2 receptor antagonists, we can mention metoclopramide, domperidone, sulpiride, and haloperidol. From a therapeutic point of view, metoclopramide and domperidone are used in gastric motility disorders, and haloperidol is used in psychotic alterations. Antagonists of DA 1 receptors are SCH23390 and clozapine. Clozapine is used for treating schizophrenia.