J Korean Soc Endocrinol.  2005 Aug;20(4):353-361. 10.3803/jkes.2005.20.4.353.

Role of Protein kinase C in Desensitization of Somatostatin-induced Calcium Signalling in NG108-15 Cells

• Kim KM
• Sung JH
• Kim MJ
• Rhie DJ
• Jo YH
• Hahn SJ
• Kim MS
• Yoon SH
• Kim BS

• KMID: 2100466
• DOI: http://doi.org/10.3803/jkes.2005.20.4.353

Abstract

BACKGROUND: Activation of G-protein coupled-somatostatin receptors induces the release of calcium from inositol 1, 4, 5-trisphosphate-sensitive intracelluar stores. G-protein-coupled receptor signaling decreases with prolonged exposure to an agonist. SEBJECTS and METHODS: Fura-2-based digital Ca2+ imaging was used to study the effects of prolonged exposure to an agonist on the somatostatin-induced intracellular Ca2+ concentration([Ca2+]i) increases in NG108-15 cells, which were differentiated with CO2-independent medium and 10micrometer forskolin.
RESULTS
Exposure to somatostatin(1micrometer) for 30 min completely desensitized the NG108-15 cells to a second somatostatin-induced response. The cells recovered gradually over 20 min following washout of the somatostatin. The desensitization was not due to depletion of the intracellular Ca2+ stores, and pretreatment for 30 min with bradykinin(100nM), which activates phospholipase C, or DADLE(D-Ala2-D-Leu5 enkephalin, 1microM), which activates phospholipase C, failed to cross-desensitize the somatostatin-evoked [Ca2+]i increases. Treatment with 8-cpt-cAMP(0.1mM) for 30min did not influence the somatostatin-induced[Ca2+]i increases. Phorbol 12, 13-dibutyrate(PdBu, 1microM) blocked the response completely. Down-regulation of PKC due to 24 h exposure of PdBu (1microM) inhibited the somatostatin-induced desensitization.
CONCLUSION
Prolonged exposure of somatostatin to NG108-15 cells desensitized the somatostatin-induced release of Ca2+ from the intracelluar store, with protein kinase C also involved in the desensitization.

MeSH Terms

Calcium*
Colforsin
Down-Regulation
Enkephalins
GTP-Binding Proteins
Inositol
Protein Kinase C*
Protein Kinases*
Somatostatin
Type C Phospholipases
Calcium
Colforsin
Enkephalins
GTP-Binding Proteins
Inositol
Protein Kinase C
Protein Kinases
Somatostatin
Type C Phospholipases

Figure

• 

  Fig. 1 Prolonged exposure to somatostatin desensitizes somatostatin-induced [Ca2+]i increase in NG108-15 cells. (A) Pseudocolor representations of [Ca2+]i were derived from fura-2-based digital images. Somatostatin-induced [Ca2+]i transients were elicited by superfusion with 1 µM somatostatin for 90s at 25 min intervals in this field. Responses from the three cells indicated by arrows in A, frame 1 are plotted in B. Somatostatin was applied as indicated by the horizontal bars. The numbers along the plot refer to the frame numbers in A. (C) Pseudocolor images show that Somatostatin responses were completely desensitized following a 30 min exposure to Somatostatin (1 µM). Responses from the three cells indicated by arrows in C, frame 1 are plotted in D. Somatostatin was applied continuously as indicated by the horizontal bar. Gaps in the recording (during which the cells were not illuminated) are indicated by diagonal lines. The numbers along the plot refer to the frame numbers in C

• 

  Fig. 2 Somatostatin-induced [Ca2+]i increases displayed a graded recovery from desensitization in NG108-15 cells. (A-D) Representative traces show recovery from 30 min exposure to somatostatin (1 µM) following wash periods of various duration. NG108-15 cells were washed with Hepes Hank's buffer (A) 2, (B) 6, (C) 10, or (D) 20 min prior to a second challenge with somatostatin (1 µM). (E) Summary plot shows the time course of recovery from desensitization for the wash times presented in A-D. Data are means +/- SE of at least 9 cells.

• 

  Fig. 3 No cross desensitization between somatostatin (SM)-and bradykinin (BK, Gq-coupled receptor agonist)-induced responses in NG108-15 cells. (A) Bradykinin (100 nM)-evoked [Ca2+]i increases were not affected by 30 min exposure to 1 µM somatostatin. (B) Somatostatin (1 µM)-evoked responses were not affected by 30 min exposure to 100 nM bradykinin.

• 

  Fig. 4 No cross desensitization between somatostatin (SM)-and DADLE (δ-opioid receptor agonist, Gi-coupled receptor agonist)-induced responses. (A) DADLE (1 µM)-evoked [Ca2+]i increases were not affected by prolonged exposure to 1 µM somatostatin. (B) Somatostatin (1 µM)-evoked responses were not affected by 30 min exposure to 1 µM DADLE.

• 

  Fig. 5 Effects of cAMP and PdBu on somatostatin-induced [Ca2+]i increase in NG108-15 cells. Cells were treated for 30 min prior to and during the second somatostatin application with (A) 0.1 mM 8-bromo-cAMP or (B) 1 µM PdBu. (C) Histogram summarizes responses normalized to initial control response for control (n=29), cAMP-treated (n=5), and PdBu-treated cells (n=7). Data are means +/- SE. *P< 0.01 relative to control and cAMP.

• 

  Fig. 6 Effects of chronic PdBu treatment on somatostatin-induced [Ca2+]i increase in NG108-15 cells. NG108-15 cells were grown in the presence of 1 µM PdBu for 24h. (A) Somatostatin (1 µM)-evoked responses were desensitized following a 30 min exposure of somatostatin (1 µM) and a 6 min wash in untreated cells. (B) Somatostatin-induced desensitization inhibited by chronic PdBu treatment. (C) Histogram summarizes responses normalized to initial control response for untreated cells (n=36) and chronic PdBu-treated cells (n= 5). *P< 0.05 relative to untreated.

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