Capacitative Ca Entry Exclusively Inhibits cAMP Synthesis in C6-2B Glioma Cells

doi:10.1074/jbc.270.3.1149

Capacitative Ca Entry Exclusively Inhibits cAMP Synthesis in C6-2B Glioma Cells

EVIDENCE THAT PHYSIOLOGICALLY EVOKED Ca ENTRY REGULATES Ca-INHIBITABLE ADENYLYL CYCLASE IN NON-EXCITABLE CELLS (*)

From the Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262

^¶ To whom correspondence should be addressed:
Dept. of Pharmacology, Box C-236, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262.
Tel.: 303-270-8964; Fax: 303-270-7097.

Abstract

Elevation of cytosolic free Ca inhibits the type VI adenylyl cyclase that predominates in C6-2B cells. However, it is not known whether there is any selective requirement for Ca entry or release for inhibition of cAMP accumulation to occur. In the present study, the effectiveness of intracellular Ca release evoked by three independent methods (thapsigargin, ionomycin, and UTP) was compared with the capacitative Ca entry that was triggered by these treatments. In each situation, only Ca entry could inhibit cAMP accumulation (La ions blocked the effect); Ca release, which was substantial in some cases, was without effect. A moderate inhibition, as was elicited by a modest degree of Ca entry, could be rendered substantial in the absence of phosphodiesterase inhibitors. Such conditions more closely mimic the physiological situation of normal cells. These results are particularly significant, in demonstrating not only that Ca entry mediates the inhibitory effects of Ca on cAMP accumulation, but also that diffuse elevations in [Ca] are ineffective in modulating cAMP synthesis. This property suggests that, as with certain Ca-sensitive ion channels, Ca-sensitive adenylyl cyclases may be functionally colocalized with Ca entry channels.

Footnotes

↵^§ Supported in part by Training Grant GM 07063 from the National Institutes of Health.
↵* The studies were supported in part by National Institutes of Health Grant GM 32483 (to D. M. F. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

[Ca]

cytosolic free Ca

G-protein

guanine nucleotide-binding regulatory protein

PDE

cAMP phosphodiesterase

TG

thapsigargin

IM

ionomycin

PTX

the toxin derived from B. pertussis, which ADP-ribosylates G-protein α, subunits

IP₃

inositol 1,4,5-trisphosphate.
²In these studies, whether isoproterenol alone, forskolin alone, or the combination was used to stimulate cAMP accumulation, the degree of inhibition associated with Ca entry was not strikingly different (data not shown). Furthermore, agents that were used to elevate cAMP were without effect on either Ca entry or release and so were not included in the [Ca] measurements.
↵³The present effects of UTP are distinct from those of ATP, which has been reported to directly inhibit adenylyl cyclase in C6-2B cells, via a PTX-sensitive G_i protein, without the involvement of Ca-influx(41, 42). Whereas ATP can bind to all purinoceptors, regardless of the signal pathways that they utilize, UTP acts selectively through the P purinoceptor, which is coupled to phospholipase C via a PTX-insensitive G_q protein in C6-2B cells(25, 40).
↵⁴Quite curiously, in this same cell line, Charpentier et al. (44) observed a prominent Ca-dependent stimulation of cAMP accumulation, which appeared to be largely due to Ca influx. The fact that we, as well as others, have demonstrated inhibition of cAMP accumulation by calcium in C6-2B cells, rather than stimulation, may be due to divergence of the cell line over time. As noted earlier, the C6-2B cells used in the present study express predominantly type VI adenylyl cyclase mRNA along with small amounts of type III(14); it is conceivable that, with time, type III might have become the dominant isoform in these other cultures.
- Received September 9, 1994.
- Revision received November 8, 1994.