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Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide.

Chrisman TD, Perkins DT, Garbers DL - Cell Commun. Signal (2003)

Bottom Line: BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum.These effects were seen within a few minutes after addition.Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, Dallas, TX, USA. David.Garbers@utsouthwestern.edu

ABSTRACT
BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s) is of significant medical importance. RESULTS: Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2), a known component of high density lipoprotein (HDL). Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P). We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor) for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5-10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK) or Akt/protein kinase B (Akt/PKB). CONCLUSION: Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this mitogenic signaling molecule likely functions as a strong adversary of the CNP/GC-B signaling pathway in the regulation of cell proliferation and other growth factor-induced phenotypes. The mechanism by which S1P desensitizes GC-B appears different than the known S1P signaling pathways.

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Related in: MedlinePlus

Sphingosylphosphorylcholine and platelet activating factor fail to desensitize GC-B. S1P, sphingosylphosphorylcholine (SPC) or platelet activating factor (PAF) were added to the final concentrations indicated. The assays for GC-B desensitisation were as described in Methods.
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Figure 7: Sphingosylphosphorylcholine and platelet activating factor fail to desensitize GC-B. S1P, sphingosylphosphorylcholine (SPC) or platelet activating factor (PAF) were added to the final concentrations indicated. The assays for GC-B desensitisation were as described in Methods.

Mentions: Lysophosphatidic acid (LPA), is also present in serum at concentrations of 2–10 μM [14], but was not found as a component of the HDL butanol extract (not shown). Both S1P and LPA are released into serum by activated platelets [15,14] and therefore represent potential regulators of tissue remodeling at sites of injury. S1P has been suggested to bind to G-protein coupled cell surface receptors (S1PR1–5) homologous to LPA receptors (LPAR1–3); it is mitogenic when added to fibroblasts and smooth muscle cells in culture [16]. S1P also has been suggested to serve as an intracellular second messenger for the mitogenic actions of serum or PDGF in fibroblasts or smooth muscle cells [17]. S1P therefore became a primary candidate as the serum and HDL desensitization factor. Sphingosine-1-phosphate rapidly desensitized GC-B (Fig 5) and at the same time induced rapid increases of pERK and pAkt in the GCB/3T3 cell line (Fig 5, inset). The S1P concentration used (100 nM) desensitized with a half-time of about 5–10 min and was maximal by 20 min prior to CNP addition. S1P was also very potent, being effective at low nM concentrations, and was much more potent than LPA (Fig 6A). The approximate 100-fold shift in the CNP concentration-response curve (Fig 6B) would effectively desensitize GC-B over the physiological concentration range for CNP (Fig 6B). In addition to S1P, we also tested two other known components of HDL, sphingosylphosphorylcholine and platelet activating factor, and neither demonstrably desensitized GC-B (Fig 7). The IC50 of 5–10 nM for S1P, similar to the nM Kd's reported for all of the S1P receptors, including Gi-promoted inhibition of adenylate cyclase [17], together with the rapid increases in pERK and pAkt (Fig 5, inset) initially suggested it acted through cell surface receptors to desensitize GC-B. The proposed intracellular pathway for S1P seems to require μM amounts for detectable responses [17,18].


Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide.

Chrisman TD, Perkins DT, Garbers DL - Cell Commun. Signal (2003)

Sphingosylphosphorylcholine and platelet activating factor fail to desensitize GC-B. S1P, sphingosylphosphorylcholine (SPC) or platelet activating factor (PAF) were added to the final concentrations indicated. The assays for GC-B desensitisation were as described in Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC305373&req=5

Figure 7: Sphingosylphosphorylcholine and platelet activating factor fail to desensitize GC-B. S1P, sphingosylphosphorylcholine (SPC) or platelet activating factor (PAF) were added to the final concentrations indicated. The assays for GC-B desensitisation were as described in Methods.
Mentions: Lysophosphatidic acid (LPA), is also present in serum at concentrations of 2–10 μM [14], but was not found as a component of the HDL butanol extract (not shown). Both S1P and LPA are released into serum by activated platelets [15,14] and therefore represent potential regulators of tissue remodeling at sites of injury. S1P has been suggested to bind to G-protein coupled cell surface receptors (S1PR1–5) homologous to LPA receptors (LPAR1–3); it is mitogenic when added to fibroblasts and smooth muscle cells in culture [16]. S1P also has been suggested to serve as an intracellular second messenger for the mitogenic actions of serum or PDGF in fibroblasts or smooth muscle cells [17]. S1P therefore became a primary candidate as the serum and HDL desensitization factor. Sphingosine-1-phosphate rapidly desensitized GC-B (Fig 5) and at the same time induced rapid increases of pERK and pAkt in the GCB/3T3 cell line (Fig 5, inset). The S1P concentration used (100 nM) desensitized with a half-time of about 5–10 min and was maximal by 20 min prior to CNP addition. S1P was also very potent, being effective at low nM concentrations, and was much more potent than LPA (Fig 6A). The approximate 100-fold shift in the CNP concentration-response curve (Fig 6B) would effectively desensitize GC-B over the physiological concentration range for CNP (Fig 6B). In addition to S1P, we also tested two other known components of HDL, sphingosylphosphorylcholine and platelet activating factor, and neither demonstrably desensitized GC-B (Fig 7). The IC50 of 5–10 nM for S1P, similar to the nM Kd's reported for all of the S1P receptors, including Gi-promoted inhibition of adenylate cyclase [17], together with the rapid increases in pERK and pAkt (Fig 5, inset) initially suggested it acted through cell surface receptors to desensitize GC-B. The proposed intracellular pathway for S1P seems to require μM amounts for detectable responses [17,18].

Bottom Line: BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum.These effects were seen within a few minutes after addition.Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, Dallas, TX, USA. David.Garbers@utsouthwestern.edu

ABSTRACT
BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s) is of significant medical importance. RESULTS: Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2), a known component of high density lipoprotein (HDL). Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P). We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor) for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5-10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK) or Akt/protein kinase B (Akt/PKB). CONCLUSION: Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this mitogenic signaling molecule likely functions as a strong adversary of the CNP/GC-B signaling pathway in the regulation of cell proliferation and other growth factor-induced phenotypes. The mechanism by which S1P desensitizes GC-B appears different than the known S1P signaling pathways.

No MeSH data available.


Related in: MedlinePlus