Involvement of Gαq/11 in m3-Muscarinic Receptor Stimulation of Phosphatidylinositol 4,5 Bisphosphate-Specific Phospholipase C in Rat Parotid Gland Membranes

doi:10.1006/abbi.1993.1459

Archives of Biochemistry and Biophysics

Volume 305, Issue 2, September 1993, Pages 546-550

https://doi.org/10.1006/abbi.1993.1459 Get rights and content

Abstract

Carbachol stimulation of phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis by rat parotid gland membranes is dependent on the presence of GTPγS and is a result of m₃-muscarinic receptor regulation of G-protein coupled, PIP₂-specific phospholipase C (PLC). The PLC activity (>80%) was solubilized with 1% Na-cholate but the solubilized enzyme was not stimulated by GTPγS and carbachol. Immunoblotting of rat parotid membranes with polyclonal antiserum, which recognizes α-subunits of the G_q/11 family, indicated the presence of two immunoreactive proteins of approximate molecular weights 41 and 42 kDa. Incubation of membranes with the common G_αq/11 antiserum attenuated the stimulation of PIP₂ hydrolysis, induced by GTPγS alone and by carbachol, in the presence of GTPγS. The antiserum had no effect on PIP₂ hydrolysis in unstimulated membranes or in the cholate extract, where it is uncoupled from the G-protein. Antiserum against G_αi, which is also coupled to the m₃-muscarinic receptor in this tissue, had no effect on either basal or stimulated PIP₂ hydrolysis. These results demonstrate that in rat parotid gland, activation of PIP₂-specific PLC by m₃-muscarinic receptor stimulation is mediated via α-subunits of the G_q/11 family of G-proteins.

References (0)

Cited by (33)

Selectivity and evolutionary divergence of metabotropic glutamate receptors for endogenous ligands and G proteins coupled to phospholipase C or TRP channels
2014, Journal of Biological Chemistry
Citation Excerpt :
Among the four types of dominant negative mutants of Gαi/o proteins (i1, i2, i3, and oA), the dominant negative mutants of Gαi2 and Gαo blocked the l-glutamate mediated mGluR1 activation of TRPC4β the most, and mutant Gαi1 or Gαi3 blocked the response to a lesser extent (Fig. 7, C–F). To test for the possibility that the dominant negative isoforms at high levels simply block activation of Gq, we titrated cells with increasing amounts of dominant-negative-expressing plasmids (Fig. 7, C–F) to reveal that mGluR1 signaling to TRPC4β can be almost completely suppressed, whereas the same level of dominant negative subunits has little effect on signaling to TRPC4β by the M3 muscarinic acetylcholine receptor, a Class A GPCR known to work through activation of Gαq/11 (45, 46). In addition, the same levels of dominant-negative Gαo that completely suppress TRPC4β activation, have no effect on Ca2+ release induced by endogenous Gq/11 in the same cell line (Fig. 8, A–B).
To define the upstream and downstream signaling specificities of metabotropic glutamate receptors (mGluR), we have examined the ability of representative mGluR of group I, II, and III to be activated by endogenous amino acids and catalyze activation of G proteins coupled to phospholipase C (PLC), or activation of G_i/o proteins coupled to the ion channel TRPC4β. Fluorescence-based assays have allowed us to observe interactions not previously reported or clearly identified. We have found that the specificity for endogenous amino acids is remarkably stringent. Even at millimolar levels, structurally similar compounds do not elicit significant activation. As reported previously, the clear exception is l-serine-O-phosphate (l-SOP), which strongly activates group III mGluR, especially mGluR4,-6,-8 but not group I or II mGluR. Whereas l-SOP cannot activate mGluR1 or mGluR2, it acts as a weak antagonist for mGluR1 and a potent antagonist for mGluR2, suggesting that co-recognition of l-glutamate and l-SOP arose early in evolution, and was followed later by divergence of group I and group II mGluR versus group III in l-SOP responses. mGluR7 has low affinity and efficacy for activation by both l-glutamate and l-SOP. Molecular docking studies suggested that residue 74 corresponding to lysine in mGluR4 and asparagine in mGluR7 might play a key role, and, indeed, mutagenesis experiments demonstrated that mutating this residue to lysine in mGluR7 enhances the potency of l-SOP. Experiments with pertussis toxin and dominant-negative Gα_i/o proteins revealed that mGluR1 couples strongly to TRPC4β through Gα_i/o, in addition to coupling to PLC through Gα_q/11.
Effects of pilocarpine on the secretory acinar cells in human submandibular glands
2006, Life Sciences
Pilocarpine has been used as a choice of drugs for treatment of impaired salivary flow. Although considerable data are available as to the stimulatory effect of pilocarpine on the salivary secretion in human, its underlying mechanism, at the cellular level, has not been rigorously studied. In this experiment, we studied the effect of pilocarpine on the ion channel activity, cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) and aquaporin (AQP)-5 expression, which play key roles in the secretary process and determine the capacity of fluid secretion. In human submandibular gland (SMG) acinar cells, 10^− 5 M pilocarpine activated the outward rectifying-current, which was predominantly K⁺ selective in the whole cell patch clamp study. The pilocarpine increased [Ca²⁺]_i in a concentration-dependent manner in the range of 10^− 6 M to 10^− 4 M. We found that both increases of [Ca²⁺]_i and outward rectifying- K⁺ current were inhibited by 10^− 5 M U-73122, a specific phospholipase C inhibitor. The magnitudes of pilocarpine-induced [Ca²⁺]_i transients were approximately 55% lower than those with the same concentration of carbachol (CCh). Pilocarpine also increased the amount of AQP-5 protein in the apical membrane (APM) in human SMG acinar cells. Our results suggest that pilocarpine induce salivary secretions in human by activating K⁺ channels, increasing [Ca²⁺]_i via phospholipase C dependent pathway, and increasing AQP-5 protein expression in the APM of SMG acinar cells.
Water channels and zymogen granules in salivary glands
2006, Journal of Pharmacological Sciences
Salivary secretion occurs in response to stimulation by neurotransmitters released from autonomic nerve endings. The molecular mechanisms underlying the secretion of water, a main component of saliva, from salivary glands are not known; the plasma membrane is a major barrier to water transport. A 28-kDa integral membrane protein, distributed in highly water-permeable tissues, was identified as a water channel protein, aquaporin (AQP). Thirteen AQPs (AQP0 – AQP12) have been identified in mammals. AQP5 is localized in lipid rafts under unstimulated conditions and translocates to the apical plasma membrane in rat parotid glands upon stimulation by muscarinic agonists. The importance of increases in intracellular calcium concentration [Ca²⁺]_i and the nitric oxide synthase and protein kinase G signaling pathway in the translocation of AQP5 is reviewed in section I. Signals generated by the activation of Ca²⁺ mobilizing receptors simultaneously trigger and regulate exocytosis. Zymogen granule exocytosis occurs under the control of essential process, stimulus-secretion coupling, in salivary glands. Ca²⁺ signaling is a principal signal in both protein and water secretion from salivary glands induced by cholinergic stimulation. On the other hand, the cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase system has a major role in zymogen granule exocytosis without significant increases in [Ca²⁺]_i. In section II, the mechanisms underlying the control of salivary protein secretion and its dysfunction are reviewed.
Spatial microenvironment defines Ca<sup>2+</sup> entry and Ca<sup>2+</sup> release in salivary gland cells
2005, Biochemical and Biophysical Research Communications
Citation Excerpt :
α-Amylase was detected in the preparation from acini but not in that from ducts, whereas kallikrein was found only in ducts. Previously, acinar cells were reported to express the M3 muscarinic receptor [10,11], but the muscarinic receptor subtype in duct cells has been unknown. The muscarinic receptor subtypes were examined in the acinar and duct cells.
The difference of Ca²⁺ mobilization induced by muscarinic receptor activation between parotid acinar and duct cells was examined. Oxotremorine, a muscarinic–cholinergic agonist, induced intracellular Ca²⁺ release and extracellular Ca²⁺ entry through store-operated Ca²⁺ entry (SOC) and non-SOC channels in acinar cells, but it activated only Ca²⁺ entry from non-SOC channels in duct cells. RT-PCR experiments showed that both types of cells expressed the same muscarinic receptor, M3. Given that ATP activated the intracellular Ca²⁺ stores, the machinery for intracellular Ca²⁺ release was intact in the duct cells. By immunocytochemical experiments, IP₃R2 colocalized with M3 receptors in the plasma membrane area of acinar cells; in duct cells, IP₃R2 resided in the region on the opposite side of the M3 receptors. On the other hand, purinergic P2Y2 receptors were found in the apical area of duct cells where they colocalized with IP₃R2. These results suggest that the expression of the IP₃Rs near G-protein-coupled receptors is necessary for the activation of intracellular Ca²⁺ stores. Therefore, the microenvironment probably affects intracellular Ca²⁺ release and Ca²⁺ entry.
cAMP potentiates ATP-evoked calcium signaling in human parotid acinar cells
2004, Journal of Biological Chemistry
In salivary acinar cells, intracellular calcium ([Ca²⁺]_i) signaling plays an important role in eliciting fluid secretion through the activation of Ca²⁺-activated ionic conductances. Ca²⁺ and cAMP have synergistic effects on fluid secretion such that peak secretion is elicited following activation of both parasympathetic and sympathetic pathways. We have recently demonstrated that cAMP exerts effects on Ca²⁺ release, through protein kinase A (PKA)-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP₃R) in mouse parotid acinar cells. To extend these findings, in the present study cross-talk between Ca²⁺ signaling and cAMP pathways in human parotid acinar cells was investigated. In human parotid acinar cells, carbachol stimulation evoked increases in the [Ca²⁺]_i and the initial peak amplitude was enhanced following PKA activation, consistent with reports from mouse parotid. Stimulation with ATP also evoked an increase in [Ca²⁺]_i. The ATP-evoked Ca²⁺ elevation was largely dependent on extracellular Ca²⁺, suggesting the involvement of the P2X family of purinergic receptors. Pharmacological elevation of cAMP resulted in a ∼5-fold increase in the peak [Ca²⁺]_i change evoked by ATP stimulation. This enhanced [Ca²⁺]_i increase was not dependent on intracellular release from InsP₃R or ryanodine receptors, suggesting a direct effect on P2XR. Reverse transcription-polymerase chain reaction and Western blot analysis confirmed the presence of P2X₄R and P2X₇R mRNA and protein in human parotid acinar cells. ATP-activated cation currents were studied using whole cell patch clamp techniques in HEK-293 cells, a null background for P2XR. Raising cAMP resulted in a ∼4.5-fold enhancement of ATP-activated current in HEK-293 cells transfected with P2X₄R DNA but had no effects on currents in cells expressing P2X₇R. These data indicate that in human parotid acinar cells, in addition to modulation of Ca²⁺ release, Ca²⁺ influx through P2X₄R may constitute a further locus for the synergistic effects of Ca²⁺ and PKA activation.
Effect of SNI-2011 on amylase secretion from parotid tissue in rats and in neuronal nitric oxide synthase knockout mice
2003, European Journal of Pharmacology
The effect of (±)cis-2-methylspilo(1,3-oxathiolane-5,3′)quinuclidine (SNI-2011) on the secretory pathway of amylase in parotid tissues was investigated. SNI-2011-induced exocytosis was inhibited by a cell-permeable Ca²⁺ chelator or inhibitors of calmodulin kinase II, neuronal nitric oxide synthase (nNOS), soluble guanyl cyclase, cyclic GMP-dependent protein kinase (PKG), and myosin light chain kinase, suggesting that these enzymes were coupled with the exocytosis. Stimulation with SNI-2011 of isolated rat parotid acinar cells loaded with 4,5-diaminofluorescein/diacetate (DAF-2/DA) induced a fast increase in DAF fluorescence corresponding to an increase in the NO production. SNI-2011-induced amylase secretion from parotid tissues in nNOS knockout mice has not been observed yet in spite of the expression of muscarinic M₃ receptors and the maintenance of secretory response to isoproterenol in the tissues. These results indicate the implication of the activation of Ca²⁺- and calmodulin-dependent enzymes and NOS-PKG signaling pathway in SNI-2011-induced amylase secretion from parotid acinar cells.

View all citing articles on Scopus

View full text

Regular ArticleInvolvement of Gαq/11 in m3-Muscarinic Receptor Stimulation of Phosphatidylinositol 4,5 Bisphosphate-Specific Phospholipase C in Rat Parotid Gland Membranes

Abstract

Regular Article
Involvement of G_αq/11 in m₃-Muscarinic Receptor Stimulation of Phosphatidylinositol 4,5 Bisphosphate-Specific Phospholipase C in Rat Parotid Gland Membranes