Markers of Adenosine Removal in Normotensive and Hypertensive Rat Nervous Tissue

Abstract

Adenosine mechanisms are altered in brain stem nuclei associated with cardiovascular control in spontaneously hypertensive rats (SHR). Therefore, in the present study we used a number of techniques to compare the binding of the adenosine transport inhibitor [ 3 H]nitrobenzylthioinosine ([ 3 H]NBMPR) as well as adenosine deaminase immunoreactivity (ADA-IR) in brain stems and nodose ganglia of SHR and age-matched normotensive Donryu rats (DRY). Saturation binding revealed a single class of [ 3 H]NBMPR binding sites in the dorsal brain stem of both strains, with K d and B max values of 65±9 pmol/L and 282±31 fmol/mg protein, respectively, in SHR and 129±2 pmol/L and 217±23 fmol/mg protein in DRY. The K d for [ 3 H]NBMPR was significantly lower in SHR than in DRY. In competition assays, NBMPR, dilazep, dipyridamole, and adenosine displaced [ 3 H]NBMPR binding, with K d values of 0.21±0.04, 57.16±16.20, 1340±100, and 87 000±12 500 nmol/L, respectively, in DRY and 0.17±0.04, 28.24±3.60, 621±100, and 32 000±6820 in SHR. K d values for all displacers were lower in SHR; however, only values for dipyridamole and adenosine reached statistical significance. Autoradiography of adenosine transport sites with [ 3 H]NBMPR revealed that unilateral nodose ganglionectomy reduced [ 3 H]NBMPR binding on the denervated side of the nucleus tractus solitarius by 20.6±1.1% in DRY and 18.7±2.3% in SHR. The density of [ 3 H]NBMPR binding in nodose ganglia was significantly lower in SHR (0.99±0.06 Bq/mm 2 ) than in DRY (1.25±0.08). Immunohistochemical studies demonstrated ADA-IR in the dorsal vagal complex, associated with both nerve cells and fibers. Measurement of ADA-IR in the dorsal vagal complex with an 125 I-labeled secondary antibody revealed a significantly higher level of ADA-IR in SHR (122%) than in DRY. In the nodose ganglia, ADA-IR was associated with a population of vagal perikarya. The present study helps provide a molecular explanation for the previously reported impaired cardiovascular responses to intra–nucleus tractus solitarius microinjection of adenosine in hypertensive rats.