In the rat proximal tubule, the alpha(2B)-adrenergic receptor (alpha(2B)-AR) enhances Na(+) reabsorption by increasing the activity of Na(+)/H(+) exchanger isoform NHE3. The mechanisms involved are unclear, and inhibition of cAMP production remains controversial. In this study, we reinvestigated alpha(2B)-AR signaling pathways using rat proximal tubule cells (PTC) in primary culture and LLC-PK(1) cells permanently transfected with the RNG gene (rat nonglycosylated alpha(2)-AR). Binding experiments indicated that PTC express substantial amounts of alpha(2B)-AR (130 fmol/mg protein), and only RNG transcripts were detected. In both cell types, the alpha(2B)-AR is coupled to G protein, and its stimulation by dexmedetomidine, but not by UK-14304, provoked a significant inhibition of the accumulation of cAMP induced by forskolin or parathyroid hormone. Exposure to alpha(2)-agonists increased arachidonic acid release and caused extracellular signal-regulated kinase (ERK)1/2 phosphorylation, which correlated with enhanced mitogen-activated protein kinse (MAPK) activity and nuclear translocation. MAPK phosphorylation was blunted by pertussis toxin but not by protein kinase C desensitization, and it coincided with transient phosphorylation of Shc. Finally, treatment with UK-14304 accelerated cell growth. Further studies will be necessary to clarify the precise mechanism of MAPK activation, but the present data suggest that alpha(2B)-AR may play a positive role during tubular regeneration.