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TRPC3 is a nonselective cation channel member of the "canonical" transient receptor potential (TRPC) family whose members are activated by phospholipase C-coupled receptors. TRPC3 can be activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) in a protein kinase C-independent manner. On the other hand, phorbol 12-myristate 13-acetate (PMA) inhibits OAG-mediated TRPC3 channel activation, suggesting that phosphorylation of TRPC3 by protein kinase C is a mechanism of receptor-mediated negative feedback. Here, we show PMA-induced phosphorylation of TRPC3 channels in vivo. We demonstrate by site-directed mutagenesis that a single site containing Ser712 and conserved among all members of the TRPC family is essential for protein kinase C-mediated negative regulation of TRPC3. In human embryonic kidney 293 cells expressing a TRPC3 mutant in which Ser712 was replaced by alanine (S712A), PMA failed to block channel activation, whereas wild-type TRPC3 activity was completely inhibited. Phosphorylation of the S712A TRPC3 mutant was not stimulated in response to PMA treatment. Furthermore, S712A TRPC3 mutant-mediated Ca2+ entry after methacholine activation was significantly greater than that of wild-type TRPC3. These findings demonstrate a dual role for phospholipase C-generated diacylglycerol, which serves as a signal for TRPC3 activation as well as a signal for negative feedback via protein kinase C-mediated phosphorylation.

We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 μm) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+from intracellular stores and influx of Ca2+from the extracellular space. A lower concentration of carbachol (5 μm) induced repetitive [Ca2+]ispikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+with 100 μmcarbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 μmGd3+, as well as 30–100 μmconcentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+entry associated with [Ca2+]ioscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+and was unaffected by 100 μm2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2+]ioscillations was insensitive to inhibition by Gd3+but was completely blocked by 100 μm2-APB. These findings indicate that the signaling pathway responsible for the Ca2+entry driving [Ca2+]ioscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2and arachidonic acid.

The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca2+, Mg2+, and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca2+stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca2+, and (ii) both the store-operated current and the single-channel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular Mg2+present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science283, 836–839), and in RBL-2H3 m1 cells a current with similar properties is activated by store depletion.

We examined the activation and regulation of calcium release-activated calcium current (Icrac) in RBL-1 cells in response to various Ca2+store-depleting agents. With [Ca2+] istrongly buffered to 100 nm,Icracwas activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP3), and two metabolically stable IP3receptor agonists, adenophostin A andl-α-glycerophospho-d-myoinositol-4,5-bisphosphate (GPIP2). With minimal [Ca2+] ibuffering, with [Ca2+] ifree to fluctuateIcracwas activated by ionomycin, thapsigargin, and by the potent IP3receptor agonist, adenophostin A, but not by GPIP2or IP3itself. Likewise, when [Ca2+] iwas strongly buffered to 500 nm,ionomycin, thapsigargin, and adenophostin A did and GPIP2and IP3did not activate detectableIcrac. However, with minimal [Ca2+] ibuffering, or with [Ca2+] ibuffered to 500 nm, GPIP2was able to fully activate detectableIcracif uptake of Ca2+intracellular stores was first inhibited. Our findings suggest that when IP3activates the IP3receptor, the resulting influx of Ca2+quickly inactivates the receptor, and Ca2+is re-accumulated at sites that regulateIcrac. Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca2+pools following activation by different IP3receptor agonists indicates that the critical regulatory pool of Ca2+may be very small in comparison to the total IP3-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP3receptors located on discrete IP3-sensitive Ca2+pools in calcium feedback regulation ofIcracand capacitative calcium entry.

Activation of a variety of cell surface receptors results in the phospholipase C‐catalyzed hydrolysis of the minor plasma membrane phospholipid phosphatidylinositol 4,5‐bisphosphate, with concomitant formation of inositol 1,4,5‐trisphosphate and diacylglycerol. There is strong evidence that inositol 1,4,5‐trisphosphate stimulates Ca2+release from intracellular stores. The Ca2+‐releasing actions of inositol 1,4,5‐trisphosphate are terminated by its metabolism through two distinct pathways. Inositol 1,4,5‐trisphosphate is dephosphorylated by a 5‐phosphatase to inositol 1,4‐bisphosphate; alternatively, inositol 1,4,5‐trisphosphate can also be phosphorylated to inositol 1,3,4,5‐tetrakisphosphate by a 3‐kinase. Although the mechanism of Ca2+mobilization is understood, the precise mechanisms involved in Ca2+entry are not known; the proposal that inositol 1,4,5‐trisphosphate secondarily elicits Ca2+entry by emptying an intracellular Ca2+pool is considered.—Putney, J. W., Jr.; Takemura, H.; Hughes, A. R.; Horstman, D. A.; Thastrup, O. Howdo inositol phosphates regulate calcium signaling? FASEB J.3: 1899‐1905; 1989.

Pemeabilised, dimethyl sulphoxide-differentiated HL-60 human myelomonocytic leukemia cells accumulate 45Ca in an ATP-dependent manner. The 45Ca is taken up by a pool thought to be a component of the endoplasmic reticulum. Inositol trisphosphate induced a rapid release of Ca from this pool, suggesting that this molecule which is formed in these cells in response to f-Met-Leu-Phe may play a role in agonist-induced Ca metabolism.

Addition of ATP (but not epinephrine, angiotensin II, vasopressin, or platelet-activating factor) to H-35 hepatoma cells whose cellular lipids have been pre-labelled with [ 3H]inositol, causes a rapid increase in [ 3H]inositol trisphosphate. In H-35 cells pre-incubated in the presence of 45Ca 2+, ATP causes a similarly rapid release of 45Ca 2+. The concentration-effect relationships for inositol trisphosphate formation and Ca 2+efflux are similar to those reported previously for differentiated hepatocytes. These results demonstrate that at least one of the Ca 2+-mobilizing receptors normally found on hepatocytes is functionally retained in the H-35 hepatoma cell line and thus could provide a useful model for the study of these receptor mechanisms in liver.

The stimulation of [3H]glucose release (a measure of glycogenolysis) from isolated guinea pig hepatocytes by Ca‐mobilizing agonists can be resolved into two phases. The initial transient phase is independent of extracellular Ca, and is probably a result of Ca released from an intracellular pool. The second phase occurs only in the presence of extracellular Ca, which suggests that Ca‐influx is also involved in the mechanism of Ca‐mobilization by these agents in the guinea pig hepatocyte.

Randriamampita and Tsien (Randriamampita, C., and Tsien, R. Y.(1993) Nature364, 809-814) suggested that an acid-extracted fraction from a Jurkat cell line contains a messenger responsible for the coupling of calcium entry to the depletion of intracellular stores, i.e.capacitative calcium entry. We found that the extract, prepared as described by Randriamampita and Tsien, caused Ca2+entry in 1321N1 astrocytoma cells which was not blocked by the D-myo-1,4,5-trisphosphate-receptor antagonist, heparin. In contrast to astrocytoma cells, when applied to mouse lacrimal acinar cells and rat hepatocytes the Jurkat extract always caused the release of intracellular Ca2+, followed by Ca2+entry across the plasma membrane. This activity of the extract on lacrimal cells was blocked by either intracellular injection of heparin or extracellular atropine. Similarly prepared lacrimal cell extracts gave Ca2+responses when applied to astrocytoma cells or lacrimal cells which were similar to those for Jurkat-derived extract. However, extracts from hepatocytes had no effect. In most Xenopusoocytes, the Jurkat extract had no effect, while in a few oocytes, the extract gave a [Ca2+]iresponse similar to that seen in lacrimal cells, that is, release of Ca2+followed by Ca2+entry. We conclude that the actions of the Jurkat cell extract are not consistent with its containing the long sought messenger for capacitative calcium entry. It is likely that this fraction contains a number of factors that mediate Ca2+response in different cell types, possibly through receptor-mediated mechanisms.

The increase in cytosolic [Ca2+] induced by Ca-mobilizing hormones in liver is mainly due to release of Ca from intracellular stores1–7. For Ca to be released from internal sites a messenger must be formed at the plasma membrane which diffuses into the cytosol to signal Ca release from the intracellular organelles2,3,7,8. One of the first actions of these hormones is to cause breakdown of the polyphosphoinositides to form soluble inositol phosphates9–11. Some evidence for the idea that these substances could be the second messenger10has been obtained in pancreatic acinar cells12. Here we have found that hormone activation of hepatocytes causes rapid breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to form inositol trisphosphate (InsP3). When applied to permeabilized hepatocytes, InsP3releases Ca from non-mitochondrial ATP-dependent pools. This suggests that InsP3could be the messenger linking Ca-mobilizing receptor activation to intracellular Ca release in liver.

The effects of receptor stimulation on net fluxes of45Ca in parotid acinar cells were investigated. When cellular45Ca content was near steady-state, muscarinic receptor activation by carbachol caused a net efflux of45Ca (not always reproducible) followed by a net influx. In the presence of excess ethylene glycol bis (ß-aminoethyl ether) N,N,N',N'-tetraacetic acid, net efflux invariably occurred, but the influx phase was prevented. When the muscarinic receptor antagonist atropine was added to the medium during the influx phase, an abrupt transient influx occured followed by a return of net influx to the prestimulation level. When cellular responses believed to reflect intracellular ionized Ca (K+ permeability, protein secretion) were examined under similar conditions, atropine invariably had an inhibitory effect. The Ca taken up in response to atropine apparently replenishes the hormone-sensitive pool of cellular Ca since it can be released subsequently by adrenoceptor activation. Taken together, these observations suggest that when atropine is administered to cholinergically activated cells, the hormone-sensitive Ca pool rapidly refills from the extracellular fluid without a concomitant increase in ionized intracellular Ca. Thus, it is suggested that this Ca pool is most likely associated with the plasma membrane.

The effects of carbachol on uptake of22Na by enzymatically dispersed rat pancreatic acinar cells were determined. Carbachol caused a slight but significant increase in uptake of22Na by the cells in the presence or absence of ouabain (10-3 M). A maximal response was obtained with 10-6 M carbachol. The effects of carbachol were blocked by 10-5 M atropine. Caerulein (10-7 M) also stimulated22Na uptake, while epinephrine (10-4 M) and substance P (10-7 M) did not. Carbachol did not stimulate22Na uptake in the absence of extracellular Ca, although Ca omission significantly clevated basal22Na uptake. The divalent cationophore A-23187 caused Ca-dependent22Na uptake at 20 µM concentration but not at 0.3 µM. These results, when considered with earlier reports by others, suggest that muscarinic receptor activation leads to an increase in permeability of the acinar cell membrane to Na, and that Ca may be second messenger for this effect.