Abstract
We previously demonstrated that the histamine H2 receptor can activate both adenylate cyclase (AC) and phospholipase C (PLC) signaling pathways via separate GTP- dependent mechanisms. We examined whether H2 receptor-specific peptides corresponding to the amino (N) or carboxyl terminus (C) of the second (2i) or third (3i) intracytoplasmic loops or the carboxyl terminal tail (P4iN) could effect histamine- stimulated AC and PLC activity in cell membranes prepared from HEPA cells stably transfected to express the canine H2 histamine receptor cDNA. Tiotidine binding and basal signaling were not altered by the synthetic peptides. H2P2iN, H2P2iC, H2P3iN and H2P4iN did not effect histamine stimulated AC activity although H2P3iC (10−4 M) significantly inhibited this parameter (65.6 ± 7.2% of maximal stimulation) (n = 6). Combination of the five peptides (H2P2iN, H2P2iC, H2P3iN, H2P3iC and H2P4iN) abolished histamine stimulated AC activity. Although all of the peptides inhibited histamine-stimulated PLC activity to a moderate degree individually, H2P3iC (10−4 M) had the greatest effect, decreasing PLC activation to 20.8 ± 6.3% of maximal stimulation (IC50 = 7.5 × 10−7 M) (n = 6). H2P3iC and the peptide combination did not alter, forskolin, GTPγs or epinephrine-stimulated AC activity nor GTPγs and vasopressin-stimulated PLC. These studies demonstrate that both the second and third intracytoplasmic loops of the histamine H2 receptor are linked to separate signaling pathways in a differential manner.
Footnotes
-
Send reprint requests to: Dr. John Del Valle, Division of Gastroenterology, University of Michigan, MSRB-I, Box 0682, Ann Arbor, MI 48109.
-
↵1 This work was supported by the National Institutes of Health (NIH Grant RO1DK47434 and funds from the University of Michigan Gastrointestinal Peptide Research Center (NIH Grant P30DK34933). I.G. is the recipient of a VA Merit Award.
- Abbreviations:
- AC
- adenylate cyclase
- PLC
- phospholipase C
- N
- amino
- C
- carboxyl
- G-protein
- guanine nucleotide binding protein
- BSA
- bovine serum albumin
- DTT
- dithiotreitol
- EBSS. Earle’s balanced salt solution
- HPLC, high-performance liquid chromatography
- AVP
- vasopressin
- AT2R1
- angiotensin II type 1 receptor
- PAFR
- platelet-activating factor receptor
- PI
- phosphoinositide
- Received September 10, 1997.
- Accepted January 16, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|