Is Insulin a stimulus for carotid body activation?

MJ Ribeiro1, C Gonzalez0,2, MP Guarino1, EC Monteiro1, SV Conde1. 1CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Pharmacology, 1169-056 Lisboa, Portugal, 2Facultad Medicina, Universidad Valladolid, IBGM, CSIC, Bioquimica y Biologia Molecular y Fisiologia, 47005 Valladolid, Spain

Introduction and aims: Carotid bodies (CB) are major peripheral chemoreceptor organs that sense changes in blood O2, CO2 and pH levels. When activated, CB releases neurotransmitters that stimulate the carotid sinus nerve (CSN), inducing reflex ventilatory compensatory responses and increasing the activity of the sympathetic nervous system (Gonzalez et al., 1994, Physiol Rev, 74, 829-898). CB is a polimodal organ, that sense several stimuli and evidence has been obtained supporting a role for the CB in glucose sensing and homeostasis (Pardal and López-Barneo 2002, Nat Neurosci, 5,197–198). However, the lack of effect of hypoglycemia on CSN activity as well as in the release of ATP and catecholamines (CA) (Bin-Jaliah et al., 2004, J Physiol, 556, 255-266; Conde et al., 2007 J Physiol, 585, 721-730), deny that hypoglycemia per se is a stimulus for CB activation. Also, Bin-Jaliah et al. (2004, J Physiol, 556, 255-266) showed that intravenous infusion of insulin increased spontaneous ventilation an effect that was absent in bilateral CSN sectioned animals. Therefore, herein we have investigated whether insulin is a stimulus for CB activation. Methods: Wistar rats (200-350g) of both sexes were used. The effect of insulin and the combined effect of insulin/hypoxia on rat CB function was assessed: in vitro by measuring CB release of CA and ATP in the whole CB and by monitoring intracellular calcium ([Ca2+]i) in dissociated CB chemoreceptor cells; and in vivo by measuring spontaneous ventilation before and after CSN cut. CB CA and ATP release were determined by HPLC and by a bioluminescence assay respectively. [Ca2+]i was determined by Ca2+ imaging with Fura-2. For in vivo experiments, animals were anaesthetized with i.p. sodium pentobarbital (60mg/kg) and were kept under 37.5 ±-0.5 ºC during experimental procedure. Respiratory frequency and tidal volume were recorded with a pneumotachometer before and after intracarotid administration of an insulin bolus (5-200 mU/Kg). These experiments were performed in euglycemic conditions to avoid effects of systemic hypoglycemia. Additionally, the presence of insulin receptor and its phosphorylation in response to insulin (1-100 nM) were evaluated by Western Blot. Animal studies were performed in agreement with the directives of the European Union (Portuguese law no. 1005/92). One and Two-Way ANOVA were used for statistical analysis. Results: We have found that insulin receptors were present in the rat CB and that insulin (1nM) increases its phosphorylation by 107% (n=3; p<0.05). Insulin (0.01 - 100nM) induces a dose-dependent release of ATP from rat whole CB with an EC50 of 0.55 nM and an Emax of 257.9 ± 31.9%; the Emax of insulin in CB ATP release being similar to the release of ATP induced by 5% O2 (251.3 ± 54.9 % increase over basal). Also, 1nM of insulin increased in 5.5 ± 0.5 ΔRI the [Ca2+]i cumulative fluorescence in dissociated CB chemoreceptor cells (n = 48, p<0.001) and potentiated the [Ca2+]i cumulative fluorescence in response to hypoxia (n = 48, p<0.001). Additionally, intracarotid administration of insulin increased basal ventilation in a dose-dependent manner, an effect abolished by CSN cut.

Conclusion: The present results support the role of insulin as a stimulus for CB activation.

Supported by FCT – PTDC/SAU-ORG/111417/2009 and 2009 L’Oreal Medals of Honor for Women in Science.