Abstract
Studies have suggested that corticotropin-releasing hormone (CRH) originating within the hypothalamic paraventricular nucleus (PVN) acts in the nucleus of the solitary tract (NTS) to regulate sympathetic and cardiovascular functions, but the anatomical distributions and cellular actions of CRH receptors (CRHRs) in the NTS are not fully understood. We conducted in situ hybridization for CRH type 1 and type 2 receptors (CRHR1 and CRHR2) in Crl:CD(SD) rats and present mRNA levels as the total pixel gray values of positive staining calculated by ImageJ. The mRNA level of CRHR2 was significantly higher than CRHR1 (1.97 ± 0.1 x 10 7 , n=4 vs. 4.56 ± 0.92 x 10 6 , n=4; p < 0.0001) in the caudal NTS (13.68 mm - 14.76 mm caudal to bregma). We then investigated how the NTS CRHRs and CRHRs signaling pathways were influenced by 7 days exposure to intermittent hypoxia (IH), a model of the arterial hypoxemias that occur during sleep apnea. IH significantly decreased CRHR2 mRNA level in caudal NTS by 34 ± 14% (1.97 ± 0.1 x 10 7 , n=4 vs. 1.3 ± 0.22 x 10 7 , n=3; P < 0.05) but did not affect CRHR1 mRNA level. To investigate the cellular actions of CRHRs in the NTS, we performed calcium imaging on NTS slice preparations using Fura-2-acetoxymethyl ester and present intracellular calcium levels as the ratio of fluorescent intensity excited by 340nm vs. 380nm. CRH induced a transient increase of intracellular calcium level (n=19) that was abolished by the voltage-dependent calcium channel blocker nifedipine. Calcium influx was attenuated by the CRHR2 antagonist K41498 (0.06 ± 0.006, n=19 vs. 0.041 ± 0.004, n=11; P < 0.01) but not by CRHR1 antagonist NBI-35965 (n=5). The CRHR2 agonist Urocortin-II induced calcium influx (0.05 ± 0.005, n=9) but not the CRHR1 agonist Stressin-I. IH also attenuated the CRH-induced calcium influx in the NTS (0.06 ± 0.006, n=19 vs. 0.04 ± 0.004, n=19; P < 0.01). Collectively, these results demonstrate that CRHR2 mediates calcium influx in NTS neurons and IH diminishes this effect by down-regulating CRHR2.
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