The dopamine D1 receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium

Background Dopamine receptors comprise two subgroups, Gs protein-coupled “D1-like” receptors (D1, D5) and Gi-coupled “D2-like” receptors (D2, D3, D4). In airways, both dopamine D1 and D2 receptors are expressed on airway smooth muscle and regulate airway smooth muscle force. However, functional expression of the dopamine D1 receptor has never been identified on airway epithelium. Activation of Gs-coupled receptors stimulate adenylyl cyclase leading to cyclic AMP (cAMP) production, which is known to induce mucus overproduction through the cAMP response element binding protein (CREB) in airway epithelial cells. We questioned whether the dopamine D1 receptor is expressed on airway epithelium, and whether it promotes CREB phosphorylation and MUC5AC expression. Methods We evaluated the protein expression of the dopamine D1 receptor on native human airway epithelium and three sources of cultured human airway epithelial cells including primary cultured airway epithelial cells, the bronchial epithelial cell line (16HBE14o-), and the pulmonary mucoepidermoid carcinoma cell line (NCI-H292) using immunohistochemistry and immunoblotting. To characterize the stimulation of cAMP through the dopamine D1 receptor, 16HBE14o- cells and NCI-H292 cells were treated with dopamine or the dopamine D1 receptor agonists (SKF38393 or A68930) before cAMP measurements. The phosphorylation of CREB by A68930 in both 16HBE14o- and NCI-H292 cells was measured by immunoblot. The effect of dopamine or A68930 on the expression of MUC5AC mRNA and protein in NCI-H292 cells was evaluated by real-time PCR and immunofluorescence staining, respectively. Results The dopamine D1 receptor protein was detected in native human airway epithelium and three sources of cultured human airway epithelial cells. Dopamine or the dopamine D1-like receptor agonists stimulated cAMP production in 16HBE14o- cells and NCI-H292 cells, which was reversed by the selective dopamine D1-like receptor antagonists (SCH23390 or SCH39166). A68930 significantly increased phosphorylation of CREB in both 16HBE14o- and NCI-H292 cells, which was attenuated by the inhibitors of PKA (H89) and MEK (U0126). Expression of MUC5AC mRNA and protein were also increased by either dopamine or A68930 in NCI-H292 cells. Conclusions These results suggest that the activation of the dopamine D1 receptor on human airway epithelium could induce mucus overproduction, which could worsen airway obstructive symptoms

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

Background: Dopamine signaling is mediated by Gs protein-coupled “D1-like” receptors (D1 and D5) and Gi-coupled “D2-like” receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling. Methods: The mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576. Results: Messenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D1-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D1-like receptor antagonists SCH23390 or SCH39166. A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576. Conclusions: These results demonstrate that the dopamine D1 receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM

Local anesthetics worsen renal function after ischemia-reperfusion injury in rats

. Local anesthetics worsen renal function after ischemia-reperfusion injury in rats. Am J Physiol Renal Physiol 286: F111-F119, 2004. First published September 30, 2003 10.1152 10. /ajprenal.00108.2003ics are widely used during the perioperative period, even in patients with preexisting renal disease. However, local anesthestics have been shown to cause cell death in multiple cell lines, including human kidney proximal tubule cells. We questioned whether local anesthetics potentiate renal dysfunction after ischemia-reperfusion (I/R) injury in vivo. Rats were implanted with subcutaneous miniosmotic pumps that continuously delivered lidocaine (2 mg⅐kg Ϫ1 ⅐h Ϫ1 ), bupivacaine (0.4 mg⅐kg Ϫ1 ⅐h Ϫ1 ), tetracaine (1 mg⅐kg Ϫ1 ⅐h Ϫ1 ), or saline vehicle, and 6 h later the rats were subjected to 30 min of renal ischemia or to sham operation. Renal function was assessed by measurement of plasma creatinine at 24 and 48 h after renal I/R injury in the presence or absence of chronic infusions of local anesthetics and correlated to histological changes indicative of necrosis. The degree of renal apoptosis was assessed by three methods: 1) DNA fragmentation detected by terminal deoxynucleotidyl transferase biotin-dUTP nickend labeling staining, 2) DNA laddering detected after agarose gel electrophoresis, and 3) morphological identification of apoptotic tubules at the corticomedullary junction. We also measured the expression of the proinflammatory markers ICAM-1 and TNF-␣. Continuous local anesthetic infusion with renal I/R injury resulted in an increased magnitude and duration of renal dysfunction compared with the saline-infused I/R group. Additionally, both apoptotic and necrotic renal cell death as well as inflammatory changes were significantly potentiated in local anesthetic-treated rat kidneys. Local anesthetic infusion alone without I/R injury had no effect on renal function. We conclude that local anesthetics potentiated renal injury after I/R by increasing necrosis, apoptosis, and inflammation. acute renal failure; apoptosis; bupivacaine; inflammation; lidocaine; necrosis; tetracaine ACUTE RENAL FAILURE (ARF) secondary to ischemia-reperfusion (I/R) injury continues to be a significant clinical problem Patients with impaired preoperative renal function undergoing aortovascular surgery are at greatest risk for developing perioperative ARF (26). Local anesthetics are widely used in clinical practice, even in patients with impaired preoperative renal function. Epidural infusions of local anesthetic are routinely used for intraoperative and postoperative analgesia (frequently lasting several days) in patients undergoing major abdominal and vascular procedures. During induction of general anesthesia for endotracheal intubation, intravenous lidocaine is given routinely to blunt the sympathetic reflex to direct laryngoscopy. Local anesthetics are used to provide surgical anesthesia and analgesia in peripheral and central nervous system nerve blocks (spinal and epidural anesthesia). In the intensive care unit, lidocaine is frequently used as an antiarrythmic agent. Several in vitro studies found that local anesthetics increase cell death via apoptosis in neuronal, lymphocytic, and osteoblastic cell lines MATERIALS AND METHODS Implantation of Miniosmotic Pumps and Renal I/R Injury All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Adult male Sprague-Dawley rats (225-275 g, Harlan Sprague-Dawley, Indianapolis, IN) were used. They had free access to rodent chow and water. Rats were anesthetized with intraperitoneal (ip) pentobarbital sodium (50 mg/kg or to effect) and implanted with subcutaneous miniosmotic pumps (model 2ML1, Alzet) that continuously delivered 10 l/h of 5% lidocaine (2 mg⅐kg Ϫ1 ⅐h Ϫ1 ), 1% bupivacaine (0.4 mg⅐kg Ϫ1 ⅐h Ϫ1 ), 2.5% tetracaine (1 mg⅐kg Ϫ1 ⅐h Ϫ1 ), or saline vehicle. The doses of local anesthetics delivered mimicked clinically administered doses for continuous epidural infusion for a 70-kg person during and after abdominal and vascular surgical procedures. Some rats were infused with 0.5% bupivacaine instead of 1% bupivacaine. Six hours later (the time required for osmotic pump priming), rats were reanesthetized with pentobarbital sodium. After 500 U of heparin were given ip, rats were placed on an electric heating pad under a warming light. Body temperature was monitored with a rectal probe and maintained at 37°C. They were allowed to spontaneously breath room air. After a laparotomy, rats were subjected to 30-min left renal ischemia after right nephrectomy. The duration of ischemia was shown in pilot studies to produce reversible and moderate renal dysfunction in rats. Some rats were subjected to only sham operation (anesthesia, laparotomy, and right nephrectomy) and received vehicle (saline) infusion, and others received a sham operation plus local anestheti

Propofol Directly Increases Tau Phosphorylation

In Alzheimer's disease (AD) and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A) activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of neurofibrillary pathology

Revisiting intra-arterial drug delivery for treating brain diseases or is it “déjà-vu, all over again”?

For over six decades intra-arterial (IA) drugs have been sporadically used for the treatment of lethal brain diseases. In recent years considerable advance has been made in the IA treatment of retinoblastomas, liver and locally invasive breast cancers, but relatively little progress has been made in the treatment of brain cancers. High resting blood flow and the presence of the blood-brain barrier (BBB), makes IA delivery to the brain tissue far more challenging, compared to other organs. The lack of advance in the field is also partly due to the inability to understand the complex pharmacokinetics of IA drugs as it is difficult to track drug concentrations in sub-second time frame by conventional chemical methods. The advances in optical imaging now provide unprecedented insights into the pharmacokinetics of IA drug and optical tracer delivery. Novel delivery methods, improved IA drug formulations, and optical pharmacokinetics, present us with untested paradigms in pharmacology that could lead to new therapeutic interventions for brain cancers and stroke. The object of this review is to bring into focus the current practice, problems, and the potential of IA drug delivery for treating brain diseases. A concerted effort is needed at basic sciences (pharmacology and drug imaging), and translational (drug delivery techniques and protocol development) levels by the interventional neuroradiology community to advance the field

Can We Find Better Bronchodilators to Relieve Asthma Symptoms?

Bronchodilators are the first line therapy during acute asthmatic exacerbations to reverse airway obstruction primarily by relaxing airway smooth muscle. Only three categories of bronchodilators exist in clinical practice: -adrenergic agonists, anticholinergics, and methylxanthines. Each of these categories have specific drugs dating back to the early 20th century, raising the question of whether or not we can find better bronchodilators. While caffeine, theophylline, atropine, and epinephrine were the first generations of therapeutics in each of these drug classes, there is no question that improvements have been made in the bronchodilators in each of these classes. In the following editorial, we will briefly describe new classes of potential bronchodilators including: novel PDE inhibitors, natural phytotherapeutics, bitter taste receptor ligands, and chloride channel modulators, which have the potential to be used alone or in combination with existing bronchodilators to reverse acute airway obstruction in the future

The GABAA agonist muscimol attenuates induced airway constriction in guinea pigs in vivo

GABAA channels are ubiquitously expressed on neuronal cells and act via an inward chloride current to hyperpolarize the cell membrane of mature neurons. Expression and function of GABAA channels on airway smooth muscle cells has been demonstrated in vitro. Airway smooth muscle cell membrane hyperpolarization contributes to relaxation. We hypothesized that muscimol, a selective GABAA agonist, could act on endogenous GABAA channels expressed on airway smooth muscle to attenuate induced increases in airway pressures in anesthetized guinea pigs in vivo. In an effort to localize muscimol's effect to GABAA channels expressed on airway smooth muscle, we pretreated guinea pigs with a selective GABAA antagonist (gabazine) or eliminated lung neural control from central parasympathetic, sympathetic, and nonadrenergic, noncholinergic (NANC) nerves before muscimol treatment. Pretreatment with intravenous muscimol alone attenuated intravenous histamine-, intravenous acetylcholine-, or vagal nerve-stimulated increases in peak pulmonary inflation pressure. Pretreatment with the GABAA antagonist gabazine blocked muscimol's effect. After the elimination of neural input to airway tone by central parasympathetic nerves, peripheral sympathetic nerves, and NANC nerves, intravenous muscimol retained its ability to block intravenous acetylcholine-induced increases in peak pulmonary inflation pressures. These findings demonstrate that the GABAA agonist muscimol acting specifically via GABAA channel activation attenuates airway constriction independently of neural contributions. These findings suggest that therapeutics directed at the airway smooth muscle GABAA channel may be a novel therapy for airway constriction following airway irritation and possibly more broadly in diseases such as asthma and chronic obstructive pulmonary disease