Norepinephrine Increases Cytosolic Ca2+ in Neurons of the Paraventricular Nucleus of the Hypothalamus via Multiple Signaling Pathways

Abstract

The paraventricular nucleus (PVN) of the hypothalamus is a critical cardiorespiratory integration site of central autonomic reflexes. A number of catecholaminergic neurons originating in the nucleus tractus solitarii, ventrolateral medulla, and locus coeruleus project to the PVN and are activated by hypoxia. Within the PVN, norepinephrine (NE) depolarizes neurons and enhances action potential discharge via alpha‐1 adrenergic receptor (α1‐AR) activation. It is unclear what effect, if any, NE has on cytosolic Ca2+. Here we examine the influence of NE on cytosolic Ca2+ in PVN neurons using fura‐2 Ca2+ imaging in isolated PVN cultures. PVN neurons were dissociated from 3–4 week old male Sprague‐Dawley rats and examined within 5 hours of dissociation. Cultures were immunopositive for the neuronal marker PAN and corticotropin‐releasing hormone, a neuropeptide heavily expressed in the PVN, suggesting neuronal identity. Neuronal identity was confirmed by increases in Ca2+ following elevated K+‐induced depolarization. We subsequently monitored the magnitude of basal (non‐phasic) Ca2+ elevation as well the number of responding neurons, defined as those that increased cytosolic Ca2+ by more than one standard deviation above mean vehicle response. NE (100 μM, 5 min, n = 68) induced an increase in basal cytosolic Ca2+ compared to aCSF vehicle (n = 63). This effect was mimicked by the α1‐AR agonist phenylephrine (PE, 10 μM, n = 42) but not the alpha‐2 adrenergic receptor (α2‐AR) agonist clonidine (CLON, 10 μM, n = 28). NE and PE, but not CLON, also significantly increased the number of responding neurons. To examine the mechanism of elevation of basal cytosolic Ca2+ by PE and α1‐AR activation, one or more of its secondary pathways were blocked. Blockade of voltage‐dependent Ca2+ channels with cadmium abolished the Ca2+ increase of PE (n = 60), as did blockers for phospholipase C (PLC, n = 26), protein kinase C (PKC, n = 31), and the inositol trisphosphate receptor (IP3R, n = 53). PKC, PLC and IP3R block reduced the number of responding neurons. Spontaneous (phasic) Ca2+ event frequency and amplitude were not affected by NE, PE, or CLON and were not studied further. However, with repeated phasic K+‐induced membrane depolarization, NE increased baseline Ca2+ yet decreased the depolarization‐induced peak amplitude. PE mimicked the basal increase in Ca2+ without affecting peak amplitude. CLON decreased peak amplitude without affecting basal Ca2+. Taken together, these data suggest NE increases cytosolic Ca2+ in PVN neurons via an α1‐AR mediated pathway which is co‐dependent on extracellular Ca2+ influx and intracellular Ca2+ release, possibly via a PLC‐mediated signaling cascade. This α1‐AR mediated effect does not affect depolarization induced Ca2+ entry, and is not caused by a change in spontaneous Ca2+ event frequency or amplitude.Support or Funding InformationSupport: HL 098602.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.