Somatostatin is known to modulate mesangial and tubular cell function and growth, but the somatostatin receptor (sst) subtypes responsible for these effects have not been defined. There are at least five different sst receptor subtypes (sst(1)-sst(5)). We used RT-PCR to demonstrate that normal human kidney consistently expresses mRNA for sst(1) and sst(2) (9 of 9 donors). Some donors expressed sst(4) or sst(5) mRNA, but none expressed sst(3) mRNA. Expression of sst(1) and sst(2) was further assessed by staining serial sections of normal human kidney with sst(1) and sst(2) antisera, Arachis hypogaea (AH) lectin (to define distal tubule/collecting duct cells), Phaseolus vulgaris lectin (proximal tubules), and Tamm-Horsfall protein (THP) antiserum (thick ascending limb of the loop of Henle). Specificity of antisera was demonstrated by transfection and absorption studies. Sst(2), but not sst(1), was expressed in glomeruli. Intense sst(1) and sst(2) staining localized exclusively to AH+ and THP+ tubules. Thus sst(1) and sst(2) subtype-selective analogs may be useful to beneficially modulate renal cell function in pathological conditions.
SH‐SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK‐N‐SH. It grows well in serum‐containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet‐derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF‐BB, we found that SH‐SY5Y cells specifically bind PDGF with a KD = 0.14 ± 0.06 nM and Bmax = 7.3 ± 2.3 pM. Functionality of these receptors was demonstrated by an increased [3H]‐thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF‐BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12–24‐h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [3H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170‐kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti‐phosphotyrosine antibody. Immunoprecipitation with anti‐PDGF β‐receptor antibody and visualization on a western blot with an anti‐phosphotyrosine antibody also revealed a 170‐kDa protein. Maximum phosphorylation of the 170‐kDa protein occurred after 5‐min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH‐SY5Y cells have specific receptors for PDGF‐BB that are functional, and can be modulated by gangliosides.
Vasoactive intestinal peptide receptors 1 (VPAC1) and 2 (VPAC2) have been identified in humans. Cell lines expressing only VPAC1 (HT-29) or VPAC2 (Molt-4b) were identified using real-time reverse transcriptase polymerase chain reaction. Vasoactive intestinal peptide (VIP) and related peptides, VIP Ϫ6 -28 , VIP , previously isolated from cultures of human leukocytes, were evaluated for their ability to bind to VPAC1 and VPAC2 and to increase the levels of cAMP in HT-29 and Molt-4b cells. VIP bound to membranes of HT-29 colon carcinoma cells and Molt-4b lymphoblasts with high affinity (K D ϭ 1.6 Ϯ 0.2 and 1.7 Ϯ 0.9 nM, respectively). VIP 4 -28 also demonstrated high-affinity binding (K D ϭ 1.7 Ϯ 0.2 and 1.7 Ϯ 0.7 nM in HT-29 and Molt-4b, respectively). VIP and VIP 4 -28 are potent VPAC1 agonists, inducing maximal 200-and 400-fold increases in cAMP, respectively. VIP demonstrated weak VPAC2 agonist activity, inducing a maximal 14-fold increase in cAMP. VIP 4 -28 had no VPAC2 agonist activity but demonstrated potent VPAC2 antagonist activity. VIP 4 -28 inhibited VPAC2-mediated increases in cAMP in Molt-4b cells up to 95%, but had no antagonistic effect on VPAC1. Lymphoblasts did not hydrolyze VIP 4 -28 to a form with VPAC1 antagonist activity. VIP 4 -28 thus is a lymphocyte-generated VIP fragment with potent agonist activity for VPAC1 and potent antagonist activity for VPAC2.
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