We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca 2ϩ . Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca 2ϩ . Specifically, 5 M thapsigargin or 3 M A-23187 (calcium ionophore), both of which increase intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca 2ϩ , suggesting that it is dependent on Ca 2ϩ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase C␤2 (i.e., cells that elevate [Ca 2ϩ ]i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca 2ϩ may represent independent second messenger signals downstream of taste receptors.calcium-sensitive adenylyl cyclase; capacitative entry; cross talk; taste transduction DURING THE PAST DECADE, numerous advances have been made in our understanding of taste transduction mechanisms. Most tastants of the sweet, bitter, and umami classes are thought to activate G protein-coupled taste receptors and their heterotrimeric G proteins. The G␥ 13 subunit appears to be taste specific and associated with G␤ 1 (14). This G␤ 1 ␥ 13 dimer has been shown directly to activate a phospholipase C (PLC␤ 2 ), stimulating the production of inositol 1,4,5-trisphosphate (IP 3 ) and eventually triggering an increase in cytoplasmic Ca 2ϩ (14,23,35,38). Strong support for this sequence of signaling events is derived from the pronounced taste deficit that results from genetic ablation of PLC␤ 2 (45). Although release of stored Ca 2ϩ (triggered by PLC␤ 2 activity) is essential, taste-evoked calcium transients in taste cells may also include a component of capacitative entry from the extracellular medium (28). Despite the recent emphasis on this IP 3 -mediated calcium release pathway, there is evidence that changes in cAMP and cGMP concentration occur after tastant stimulation. Cyclic nucleotide modulation has been demonstrated after stimulation with some sweet, bitter, and umami stimuli (1,39,44), and exogenously applied cAMP appears to mimic tastant-evoked activity (8). Nevertheless, the significance and source of cAMP and cGMP in taste transduction are unclear, given the essential role of...

To establish the relevance of targeting disease-associated T cells in anti-RNP-associated glomerulonephritis, mice developing nephritis following immunization with U1-70-kd small nuclear ribonucleoprotein (snRNP) were treated with a single dose of irradiated antigen-selected T cell vaccine. T cell receptor usage in nephritic kidneys revealed oligoclonal use of T Cell Receptor V Beta (TRBV) genes as previously found in spleens and lungs of immunized mice with pulmonary disease. The CDR3 regions from T cell isolates showed sequence homology to those in humans with anti-RNP autoimmunity. Following T cell vaccination, urinalysis returned to normal in 5/7 treated mice (71% response rate) whereas all mock treated mice continued to have an active urinary sediment (Fisher's Exact p=0.02). An oligoclonal population of T cells homologous to those identified in humans with anti-RNP autoimmunity is implicated in disease pathogenesis, and T cell vaccination is associated with a high rate of clinical improvement in established nephritis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with significant morbidity and mortality. We have identified and characterized U1-70kDa small nuclear ribonucleoprotein (70kDa) autoantigen specific T cells in a murine model of autoimmunity when a subset of mice developed kidney disease similar to lupus nephritis. Mice developed proteinuria and histology showed CD4+ T cells associated with glomerular lesions. CD4+ T cells were freshly isolated from kidney and spleen and cultured with autoantigen to generate short-term T cell lines. Following RNA extraction from cell lines and cDNA preparation, a PCR reaction was performed using primers for the TRBV subgroups. The product was purified and underwent cloning and sequencing. T cell epitope mapping revealed that the majority of T cells were directed against antigenic peptides residing within the RNA binding domain of 70KDa. We also found that TCR beta (TRB) V usage was highly restricted among 70kDa reactive T cells which selectively utilized TCR BV subgroups 1 and 8.3 in the kidney and 1,2, 6, 8.1, 8.2, and 8.3 in the spleen and the lung. The TRB third complementary-determining regions (CDR3) had conserved sequence motifs which were shared across different TRBV subgroups. When compared for homology, the TRBV and CDR3 regions used by both murine and human 70kDa-specific CD4+ T cells were highly similar including those CD4+ T cells found at two sites of inflammation; the kidney and lung. The NIH and Dept Veterans Affairs provided support for this work.