to 
compare the expression of DAR in sc adipose tissue, striatum and pituitary. Four of the five DAR were detected in adipose tissue, with D1R being the most abundant. As expected, the A-83-01 biological activity pituitary showed high expression of D2R, D4R and D5R, while the striatum showed differential expression of all DAR. As determined by qPCR, the relative expression of D1R is higher, while that of D2R is lower in mature adipocytes than the SVC fraction, while expression of D4R was similar in the two fractions. cAMP and cGMP determinations Cells were incubated with the various treatments for 30 min and then lysed in 0.1 M HCl. After centrifugation, the supernatant was analyzed for cAMP and cGMP using respective ELISA kits from Cayman Chemical Co. To increase assay 4 September 2011 | Volume 6 | Issue 9 | e25537 Dopamine Receptors in Human Adipocytes Western blotting was used to compare expression of the DAR proteins in primary adipocytes, LS14 and SW872 cells before and after differentiation. Fig. 1C shows that D1R, D2R and D4R proteins were expressed at variable amounts in the pituitary, and in the three types of adipocytes. There was an apparent downregulation of D1R and D4R, but upregulation of D2R, in LS14 cells after differentiation. All receptors examined showed some evidence of isoforms. Changes in D1R, D2R and D4R mRNA levels during adipogenesis in LS14 cells were determined by qPCR. Fig. 1D shows a reduction in D1R, but an increase in D2R, during the first three days of differentiation. Expression of D4R was reduced on day 3 and remained suppressed until day 10. PRL expression was increased during the first six days of differentiation, followed by a decline, whereas PRLR expression showed a significant reduction throughout adipogenesis. Adipocytes express an active ARSA and respond to DA-S We used qPCR to compare the expression of arylsulfatase A, specific for catecholamines and cerebrosides, arylsulfatase B, specific for glycosaminoglycans, and arylsulfatase C, specific for steroids. The mRNA levels of “ 21526763 ARSA and ARSC in sc adipose tissue were 4.5 and 2.5 fold higher, respectively, than those of ARSB. An enzymatic assay that measures total arylsulfatase activity was then employed. In the presence of silver nitrate, only ARSA activity is blocked, enabling the calculation of its activity by subtraction from total enzyme activity. As shown in Fig. 2B, basal ARSA activity was detectable in non-differentiated LS14 and SW872 cells, increasing 8 and 20 fold, respectively, after differentiation. ARSA activity was also detectable in CM from differentiated adipocytes. To determine whether adipocytes can convert DA-S to bioactive DA, the effects of DA and DA-S on PRL release from sc adipose explants were compared. Fig. 2C shows a similar inhibition of PRL release by both DA and DA-S. The b-AR ” agonist isoproterenol, stimulated PRL release, showing an inverted U-shaped dose-dependent curve. PRL release from all types of adipocytes is inhibited by both DA and bromocriptine We next examined whether DA directly affects PRL release by the adipocytes. Fig. 3 shows a similar DA-induced inhibition of PRL release from mature adipocytes, differentiated primary preadipocytes, differentiated LS14 cells, and differentiated SW872 cells. Bromocriptine, a specific D2R agonist, mimicked the inhibitory effect of DA. A non-monotonic dose-dependent inhibition of PRL release was apparent in all cases, with 1 and 10 nM, but not 100 nM, of DA and BRO showing effective inhibition. Both D
