- Email: [email protected]
M1, M2, and M3 muscarinic receptors critically regulate cilia-driven airway epithelial particle transport
Abstracts
vagal nerves, which approximately corresponds to “the lung area” essential to the lung ventilation. Comparing with the RCPG configuration of rats, the area activated during Bd corresponds to the ventral respiratory group including the pre-Botzinger complex, which mainly exhibits inspiratory activity, and the area activated during Bc may corresponds to the para-facial respiratory group, which shows a biphasic activity during the preinspiratory and the postinspiratory phases. We suggest that Bd and Bc of frogs correspond to inspiration and postinspiratory expiration of mammals, respectively. doi:10.1016/j.autneu.2007.06.111
I-4B-08 Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration Wolfgang Kummer a, Petra Hartmann a, Katrin S. Lips a , Simone Biallas b, Uwe Pfeil a, Sergei A. Grando c , Veronika Grau b, Zbigniew Mikulski a a Institute for Anatomy and Cell Biology, Justus-Liebig– University Giessen, Germany b Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, Justus-Liebig–University Giessen, Germany c Department Dermatol. University Calif., Sacramento, California, USA Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study we set out first to determine the nAChR inventory of alveolar macrophages (AM) in the rat, and second to investigate the cellular events evoked by stimulation of AM with nicotine. Rat AM were isolated freshly by bronchoalveolar lavage (BAL). Inter-individually variable expression patterns of nAChR subunits investigated by RTPCR were noted. Positive results were obtained for subunits alpha 3, 5, 9, 10 and beta 2. Most stable expression was noted for subunits alpha 9, alpha 10 and beta 2. Notably, mRNA coding for subunit alpha 7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. Subunits alpha 3, 5, 9 and 10 and beta 2 were also detected by immunohistochemistry on AM isolated by BAL as well as on AM in lung tissue sections. Changes in intracellular calcium concentrations were monitored in rat AM by fura-2. Nicotine (100 μM) alone had almost no effect upon intracellular [Ca2+] whereas ATP (200 μM) induced a drastic increase with rapid onset and long duration. Nicotine (1 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in intracellular [Ca2+ ] by 30%. This effect was blocked by αbungarotoxin and was not dependent on the presence of
71
extracellular calcium. These data demonstrate the presence of functionally active nAChR that interact with ATP-induced rise in intracellular [Ca2+] originating from intracellular stores in rat AM. doi:10.1016/j.autneu.2007.06.112
I-4B-09 M1, M2, and M3 muscarinic receptors critically regulate cilia-driven airway epithelial particle transport Peter Koenig a,b, Maike K. Klein b, Rainer V. Haberberger b,c, Petra Hartmann b, Katrin S. Lips b, Benjamin Krain b,d, Juergen Wess e, Wolfgang Kummer b a Institut fuer Anatomie, Universitaet zu Luebeck, Luebeck, Germany b Institut fuer Anatomie und Zellbiologie and University of Giessen Lung Center, Justus-Liebig–Universitaet Giessen, Giessen, Germany c Department of Anatomy and Histology, Flinders University, Adelaide, SA, Australia d Abteilung fuer Anaesthesie, Operative Intensivmedizin, Schmerztherapie, Justus-Liebig–Universitaet Giessen, Giessen, Germany e Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA Blockade of specific muscarinic receptor (MR) subtypes is an important therapy concept in obstructive airway diseases. However, a known side-effect of pharmacological blockade of MR is reduction of mucociliary clearance and ciliary beat frequency. The specific MR subtypes involved in this action are unknown. Using five different mouse strains deficient for M1R–M5R we found that the M3R increased particle transport speed through a direct increase of ciliary beat frequency. Loss of the M3R prevented an increase of particle transport speed in response to muscarine and also blunted the response to ATP. M2R exhibited an inhibitory role and reduced the increase in particle transport speed in response to muscarine and ATP. Consequently, additional knockout of the M2R in M3R deficient mice rescued the blunted response to ATP and even partially restored that to muscarine. In contrast to M3R deficient mice, deficiency of the M1R resulted in a reduced particle transport speed in response to muscarine and ATP although the ciliary beat frequency responses to both drugs were normal. Furthermore, the reduced particle transport speed response to ATP could not be rescued by blockade of MR. None of the investigated mutant mouse strains showed any changes in relative cell composition or cell morphology in the epithelium that could explain the observed differences in particle transport speed. Our results suggest that drugs that spare M1R function and block M2R and M3R are likely to
72
Abstracts
reduce side effects in the treatment of obstructive airway diseases. doi:10.1016/j.autneu.2007.06.113
I-4B-10 Respiratory modulation of sympathetic preganglionic neuronal activity in neonatal rats Anthony E. Pickering, Alexey O. Stalbovskiy, Julian F.R. Paton Departments of Physiology and Anaesthesia, University of Bristol, Bristol, UK Sympathetic outflows to target organs show specific patterns of respiratory modulation (Habler et al. 1994). Single unit recordings show striking differences in respiratory drive between sympathetic preganglionic neurones (SPN) (Gilbey et al. 1986). The central mechanism(s) underlying the differences in sympathetic respiratory modulation are not known. We addressed this question at the level of the SPN using whole cell recording (WCR) in the working heart– brainstem preparation (WHBP, Paton 1996).Neonatal Wistar rats (p8–12) were anaesthetised with halothane, decerebrated precollicularly and perfused with carbogenated Ringers solution (32 °C). Phrenic nerve activity was recorded and the thoracic sympathetic chain stimulated with a bipolar electrode. After laminectomy (T5–C8), SPN were located using extracellular recording. Patch electrodes were driven into this area to obtain WCR (solution containing Lucifer yellow). Respiratory drive could be either increased by stimulating peripheral chemoreceptors (NaCN, i.a.) or arrested by topical cold saline to the snout to evoke a diving response. Of 62 spinal cells, 22 were identified as SPN (3 were identified definitively and 19 were classed as putative on the basis of their electrophysiology (Dembowsky et al. 1986; Pickering et al. 1991)). SPN were either spontaneously active (n = 13) or quiescent (n = 9). Many (62%) of the spontaneously active SPN had distinct patterns of respiratory related firing (inspiratory (n = 3), expiratory (n = 4) or preinspiratory (n = 1)) whereas only 22% of the quiescent cells showed respiratory modulation of excitability. The respiratory modulation was generated by phasic bursts of either excitatory or inhibitory postsynaptic potentials. Most of the spontaneously active SPN were excited by chemoreflex activation (83%), with a shift to post-inspiratory bursting, whereas quiescent SPN were mostly not excited (89%). Diving reflex activation provoked excitation in 75% and 57% of spontaneously active and quiescent SPN respectively with only one cell inhibited. Some quiescent SPN (n = 4) showed no respiratory modulation under any condition. We have obtained WCR from SPN in a preparation with strong respiratory drive. We have observed a range of different patterns of respiratory modulation of SPN excitability. Using WCR in this in situ preparation, we can examine the relative contributions of intrinsic SPN properties
and synaptic inputs that determine the distinct patterns of sympathetic motor activity. Dembowsky K J et al. (1986). Pflugers Arch 406, 112–20.Gilbey MP et al. (1986) J Physiol 378, 253–65.Habler HJ et al. (1994) Prog Neurobiol 43, 567– 606.Paton J F (1996) J Neurosci Methods 65, 63–8.Pickering AE et al. (1991) Neurosci Lett 130, 237–42. doi:10.1016/j.autneu.2007.06.114
I-P-001 Aging process of the human splanchnic nerves: A morphometric comparison Naoko Nonaka a, Noboru Goto b, Jun Goto c a The Department of Oral Anatomy, Showa University School of Dentistry, Tokyo, Japan b Koriyama Professional Training College of Health Sciences, Koriyama, Japan c The Department of Anatomy, Showa University School of Medicine, Tokyo, Japan The aim of the present study is to analyze and compare nerve fibers of the human greater and lesser splanchnic nerves in relation to the aging process. The analysis was conducted with the use of a new fixation, embedding and staining method that makes it possible to discriminate various structures of the nervous tissue and with the help of an image analyzer. We examined 25 greater splanchnic and 30 lesser splanchnic nerves which were taken from cadavers (44–96 years) for anatomy dissection. The results showed that there was a decrease in transverse area and perimeter of axons in relation to aging process for the lesser splanchnic nerve, while there was an increase for the greater splanchnic nerve. This discrepancy may indicate a difference in sympathetic nerve controls between the kidney and abdominal organs during the aging process. doi:10.1016/j.autneu.2007.06.115
I-P-002 Characteristics of mechanoreceptive units in the rat urinary bladder Nobuyuki Ishii a, Kazuo Toda b, Toshiya Terao a, Makoto Morozumi a , Tetsuo Hayashi a, Takumi Yamada a a Department of Urology, Saitama Medical Center, Saitama Medical School, Saitama, Japan b Integrative Sensory Physiology, Graduate School of Biomedical Sciences, Nagasaki University, Japan Background and aims Sensory information from the urinary bladder is carried out mainly by the pelvic nerve and the hypogastric nerve. However little is known about the response properties of
vagal nerves, which approximately corresponds to “the lung area” essential to the lung ventilation. Comparing with the RCPG configuration of rats, the area activated during Bd corresponds to the ventral respiratory group including the pre-Botzinger complex, which mainly exhibits inspiratory activity, and the area activated during Bc may corresponds to the para-facial respiratory group, which shows a biphasic activity during the preinspiratory and the postinspiratory phases. We suggest that Bd and Bc of frogs correspond to inspiration and postinspiratory expiration of mammals, respectively. doi:10.1016/j.autneu.2007.06.111
I-4B-08 Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration Wolfgang Kummer a, Petra Hartmann a, Katrin S. Lips a , Simone Biallas b, Uwe Pfeil a, Sergei A. Grando c , Veronika Grau b, Zbigniew Mikulski a a Institute for Anatomy and Cell Biology, Justus-Liebig– University Giessen, Germany b Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, Justus-Liebig–University Giessen, Germany c Department Dermatol. University Calif., Sacramento, California, USA Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study we set out first to determine the nAChR inventory of alveolar macrophages (AM) in the rat, and second to investigate the cellular events evoked by stimulation of AM with nicotine. Rat AM were isolated freshly by bronchoalveolar lavage (BAL). Inter-individually variable expression patterns of nAChR subunits investigated by RTPCR were noted. Positive results were obtained for subunits alpha 3, 5, 9, 10 and beta 2. Most stable expression was noted for subunits alpha 9, alpha 10 and beta 2. Notably, mRNA coding for subunit alpha 7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. Subunits alpha 3, 5, 9 and 10 and beta 2 were also detected by immunohistochemistry on AM isolated by BAL as well as on AM in lung tissue sections. Changes in intracellular calcium concentrations were monitored in rat AM by fura-2. Nicotine (100 μM) alone had almost no effect upon intracellular [Ca2+] whereas ATP (200 μM) induced a drastic increase with rapid onset and long duration. Nicotine (1 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in intracellular [Ca2+ ] by 30%. This effect was blocked by αbungarotoxin and was not dependent on the presence of
71
extracellular calcium. These data demonstrate the presence of functionally active nAChR that interact with ATP-induced rise in intracellular [Ca2+] originating from intracellular stores in rat AM. doi:10.1016/j.autneu.2007.06.112
I-4B-09 M1, M2, and M3 muscarinic receptors critically regulate cilia-driven airway epithelial particle transport Peter Koenig a,b, Maike K. Klein b, Rainer V. Haberberger b,c, Petra Hartmann b, Katrin S. Lips b, Benjamin Krain b,d, Juergen Wess e, Wolfgang Kummer b a Institut fuer Anatomie, Universitaet zu Luebeck, Luebeck, Germany b Institut fuer Anatomie und Zellbiologie and University of Giessen Lung Center, Justus-Liebig–Universitaet Giessen, Giessen, Germany c Department of Anatomy and Histology, Flinders University, Adelaide, SA, Australia d Abteilung fuer Anaesthesie, Operative Intensivmedizin, Schmerztherapie, Justus-Liebig–Universitaet Giessen, Giessen, Germany e Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA Blockade of specific muscarinic receptor (MR) subtypes is an important therapy concept in obstructive airway diseases. However, a known side-effect of pharmacological blockade of MR is reduction of mucociliary clearance and ciliary beat frequency. The specific MR subtypes involved in this action are unknown. Using five different mouse strains deficient for M1R–M5R we found that the M3R increased particle transport speed through a direct increase of ciliary beat frequency. Loss of the M3R prevented an increase of particle transport speed in response to muscarine and also blunted the response to ATP. M2R exhibited an inhibitory role and reduced the increase in particle transport speed in response to muscarine and ATP. Consequently, additional knockout of the M2R in M3R deficient mice rescued the blunted response to ATP and even partially restored that to muscarine. In contrast to M3R deficient mice, deficiency of the M1R resulted in a reduced particle transport speed in response to muscarine and ATP although the ciliary beat frequency responses to both drugs were normal. Furthermore, the reduced particle transport speed response to ATP could not be rescued by blockade of MR. None of the investigated mutant mouse strains showed any changes in relative cell composition or cell morphology in the epithelium that could explain the observed differences in particle transport speed. Our results suggest that drugs that spare M1R function and block M2R and M3R are likely to
72
Abstracts
reduce side effects in the treatment of obstructive airway diseases. doi:10.1016/j.autneu.2007.06.113
I-4B-10 Respiratory modulation of sympathetic preganglionic neuronal activity in neonatal rats Anthony E. Pickering, Alexey O. Stalbovskiy, Julian F.R. Paton Departments of Physiology and Anaesthesia, University of Bristol, Bristol, UK Sympathetic outflows to target organs show specific patterns of respiratory modulation (Habler et al. 1994). Single unit recordings show striking differences in respiratory drive between sympathetic preganglionic neurones (SPN) (Gilbey et al. 1986). The central mechanism(s) underlying the differences in sympathetic respiratory modulation are not known. We addressed this question at the level of the SPN using whole cell recording (WCR) in the working heart– brainstem preparation (WHBP, Paton 1996).Neonatal Wistar rats (p8–12) were anaesthetised with halothane, decerebrated precollicularly and perfused with carbogenated Ringers solution (32 °C). Phrenic nerve activity was recorded and the thoracic sympathetic chain stimulated with a bipolar electrode. After laminectomy (T5–C8), SPN were located using extracellular recording. Patch electrodes were driven into this area to obtain WCR (solution containing Lucifer yellow). Respiratory drive could be either increased by stimulating peripheral chemoreceptors (NaCN, i.a.) or arrested by topical cold saline to the snout to evoke a diving response. Of 62 spinal cells, 22 were identified as SPN (3 were identified definitively and 19 were classed as putative on the basis of their electrophysiology (Dembowsky et al. 1986; Pickering et al. 1991)). SPN were either spontaneously active (n = 13) or quiescent (n = 9). Many (62%) of the spontaneously active SPN had distinct patterns of respiratory related firing (inspiratory (n = 3), expiratory (n = 4) or preinspiratory (n = 1)) whereas only 22% of the quiescent cells showed respiratory modulation of excitability. The respiratory modulation was generated by phasic bursts of either excitatory or inhibitory postsynaptic potentials. Most of the spontaneously active SPN were excited by chemoreflex activation (83%), with a shift to post-inspiratory bursting, whereas quiescent SPN were mostly not excited (89%). Diving reflex activation provoked excitation in 75% and 57% of spontaneously active and quiescent SPN respectively with only one cell inhibited. Some quiescent SPN (n = 4) showed no respiratory modulation under any condition. We have obtained WCR from SPN in a preparation with strong respiratory drive. We have observed a range of different patterns of respiratory modulation of SPN excitability. Using WCR in this in situ preparation, we can examine the relative contributions of intrinsic SPN properties
and synaptic inputs that determine the distinct patterns of sympathetic motor activity. Dembowsky K J et al. (1986). Pflugers Arch 406, 112–20.Gilbey MP et al. (1986) J Physiol 378, 253–65.Habler HJ et al. (1994) Prog Neurobiol 43, 567– 606.Paton J F (1996) J Neurosci Methods 65, 63–8.Pickering AE et al. (1991) Neurosci Lett 130, 237–42. doi:10.1016/j.autneu.2007.06.114
I-P-001 Aging process of the human splanchnic nerves: A morphometric comparison Naoko Nonaka a, Noboru Goto b, Jun Goto c a The Department of Oral Anatomy, Showa University School of Dentistry, Tokyo, Japan b Koriyama Professional Training College of Health Sciences, Koriyama, Japan c The Department of Anatomy, Showa University School of Medicine, Tokyo, Japan The aim of the present study is to analyze and compare nerve fibers of the human greater and lesser splanchnic nerves in relation to the aging process. The analysis was conducted with the use of a new fixation, embedding and staining method that makes it possible to discriminate various structures of the nervous tissue and with the help of an image analyzer. We examined 25 greater splanchnic and 30 lesser splanchnic nerves which were taken from cadavers (44–96 years) for anatomy dissection. The results showed that there was a decrease in transverse area and perimeter of axons in relation to aging process for the lesser splanchnic nerve, while there was an increase for the greater splanchnic nerve. This discrepancy may indicate a difference in sympathetic nerve controls between the kidney and abdominal organs during the aging process. doi:10.1016/j.autneu.2007.06.115
I-P-002 Characteristics of mechanoreceptive units in the rat urinary bladder Nobuyuki Ishii a, Kazuo Toda b, Toshiya Terao a, Makoto Morozumi a , Tetsuo Hayashi a, Takumi Yamada a a Department of Urology, Saitama Medical Center, Saitama Medical School, Saitama, Japan b Integrative Sensory Physiology, Graduate School of Biomedical Sciences, Nagasaki University, Japan Background and aims Sensory information from the urinary bladder is carried out mainly by the pelvic nerve and the hypogastric nerve. However little is known about the response properties of