β-Nicotinamide adenine dinucleotide: a novel bronchodilator inducing increase in intracellular cAMP

I Jurastow; S Engel; S Wiegand; A Rafiq; A Zakrzewicz; V Grau; C Nassenstein; M Bünemann; W Kummer

doi:10.1055/s-0035-1556611

β-Nicotinamide adenine dinucleotide: a novel bronchodilator inducing increase in intracellular cAMP

I Jurastow ¹^,², S Engel ³, S Wiegand ¹^,², A Rafiq ¹^,², A Zakrzewicz ²^,⁴, V Grau ²^,⁴, C Nassenstein ¹^,², M Bünemann ³, W Kummer ¹^,²

¹Institute of Anatomy and Cell Biology, Justus-Liebig-University, Gießen
²Universities of Gießen and Marburg Lung Center (UGMLC), German Center for Lung Research
³Department of Pharmacology and Clinical Pharmacy, University of Marburg
⁴Department of General, Visceral, Thoracic, Transplantation and Pediatric Surgery, University Hospital Gießen

Abstract

Introduction: β-Nicotinamide adenine dinucleotide (β-NAD) has been reported to relax vascular and intestinal smooth muscle via purinergic P2Y1 and P2Y11 receptors, and we recently demonstrated its relaxing activity on murine and human airway smooth muscle. This bronchodilator activity, however, was independent from purinergic receptors, and occurred concomitantly with increases in intracellular calcium concentration and cell depolarization. We here set out to further evaluate intracellular signaling pathways induced by β-NAD in airway smooth muscle.

Methods: The relaxing effect of β-NAD was investigated by force recording of precontracted (10µM muscarine) murine trachea in organ bath experiments. Intracellular cAMP was measured in cultured human bronchial smooth muscles cells (HBMSC; Promocell, Heidelberg) transfected by electroporation with a FRET-sensor based on exchange protein directly activated by cAMP (EPAC).

Results: HBMSC responded to β-NAD (1 mM) with an increase in intracellular cAMP concentration which was additionally elevated by isoproterenol (10µM), a known stimulator of membrane-bound adenylate cyclase via activation of β2-adrenoreceptors. This additive effect certainly is pointed towards independent pathways of cAMP generation. Rolipram (100µM), an inhibitor of the cAMP degrading enzyme phosphodiesterase 4, augmented β-NAD-induced relaxation of precontracted murine trachea. Relaxation was insensitive, however, to phospholipase C-inhibition by U73122, to the purinergic inhibitors suramin, PPADS, and MRS2179, to inhibition of G(s)- and G(i)-signaling by pertussis toxin and cholera toxin, respectively, to NO-synthase blockade by L-NAME, inhibition of endoplasmic reticulum Ca2+-ATPase by thapsigargin, of Rho kinase by y-27632, of PKA by rp-cAMPs, and to activation of L-type voltage dependent calcium channels by FPL 64176.

Discussion: The data demonstrate that β-NAD increases intracellular cAMP, which is likely linked to its bronchodilator effect since it is augmented by phosphodiesterase 4-inhibition. Further, there is increase in calcium concentration and depolarization of the cell membrane. β-NAD potentially represents a novel therapeutic tool inobstructive airway disorders.

*Presenting author