Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1

Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr696) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser19) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.

Pharmacological Characterization of Inositol 1,4,5-tris Phosphate Receptors in Human Platelet Membranes

The phosphatidylinositol (PI) hydrolysis signaling system has been shown to be altered in platelets of depressed and schizophrenic subjects. Inositol (1,4,5) trisphosphate (Ins(1,4,5)P3), an integral component of the PI signaling system, mobilizes Ca2+ by activating Ins(1,4,5)P3 receptors. To eventually investigate the role of Ins(1,4,5)P3 receptors in depression and other mental disorders, we characterized [H3]Ins(1,4,5)P3 binding sites in crude platelet membranes prepared from small amounts of blood obtained from healthy human control subjects. We found a single, saturable binding site for [H3]Ins(1,4,5)P3 to crude platelet membranes, which is time dependent and modulated by pH, inositol phosphates, and heparin. Since cyclic adenosine monophosphate (cAMP) and Ca2+ have been shown to be important modulators in Ins(1,4,5)P3 receptors, in the present study we also determined the effects of various concentrations of CaCI2 and forskolin on Ins(1,4,5)P3 binding to platelet membranes. CaCI2 modulated [3H]Ins(1,4,5)P3 binding sites in a biphasic manner: at lower concentrations it inhibited [3H]Ins(1,4,5)P3 binding, whereas at higher concentrations, it stimulated [3H]Ins(1,4,5)P3 binding. On the other hand, forskolin inhibited [3H]Ins(1,4,5)P3 binding. Our results thus suggest that the pharmacological characteristics of [3H]Ins(1,4,5)P3 binding to crude platelet membranes are similar to that of Ins(1,4,5)P3 receptors; and that both Ca2+ and cAMP modulate [3H]Ins(1,4,5)P3 binding in crude platelet membranes.

The effect of oxytocin on progesterone secretion, phosphoinositide hydrolysis and intracellular mobilisation of Ca 2+ in porcine luteal cells

Oxytocin (OT) is involved in the regulation of steroid secretion by the corpus luteum (CL) in pigs, but OT signal transduction in the porcine CL has not been identified. In this study, the effects of OT on in vitro progesterone (P 4 ) secretion, phosphoinositide (PI) hydrolysis and intracellular mobilisation of Ca 2+ ([Ca 2+ ] i ) were investigated in porcine luteal cells during the early (days 3–5), mid-(days 8–10) and late luteal phases (days 12–14) of the oestrous cycle. Basal concentrations of P 4 and accumulation of inositol phosphates (IPs) were higher (P < 0.05) on days 3–5 and 8–10 of the oestrous cycle than on days 12–14. Basal [Ca 2+ ] i mobilisation did not differ among studied periods of the oestrous cycle. Oxytocin (10 −7 M) enhanced P4 secretion and PI hydrolysis (P < 0.05) by luteal cells harvested on days 8–10 of the oestrous cycle. Moreover, OT started to increase mobilisation of [Ca 2+ ] i at the 15th (days 3–5 and 8–10) or 30th second (days 12–14) in porcine luteal cells. It was concluded that in pigs OT acts as a regulator of steroidogenesis, stimulating P 4 secretion in mature CL. This OT action may be mediated by changes in PI hydrolysis and [Ca 2+ ] i mobilisation.

Inhibition of Gαq-dependent PLC-β1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2

In smooth muscle of the gut, Gq-coupled receptor agonists activate preferentially PLC-β1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP3) generation and induce IP3-dependent Ca2+ release. Inhibition of Ca2+ mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP3 receptor type I. The mechanism of inhibition of PLC-β1-dependent PI hydrolysis has not been established. Neither Gq nor PLC-β1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and Gαq·GTP and intrinsic Gαq·GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:Gαq·GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or Gαq(G188S), a Gαq mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-β1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of Gαq·GTP. PKG acts only via phosphorylation of RGS4.

Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors

The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC50 0.7 ± 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated Gq and G13, stimulated Gαq-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP3)-dependent Ca2+ release, and increased cytosolic free Ca2+. PI hydrolysis was blocked by expression of Gαq minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC50 = 1.0 ± 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca2+-dependent contraction and myosin light-chain (MLC)20 phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC20 phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of Gαq or Gα13 minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC20 phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a Gαq-mediated cascade involving Ca2+/calmodulin activation of MLC kinase and transient MLC20 phosphorylation and contraction as well as a sustained Gαq- and Gα13-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC20 phosphorylation and contraction.

Activation of PLC-δ1 by Gi/o-coupled receptor agonists

The mechanism of phospholipase (PLC)-δ activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca2+ stimulated an eightfold increase in PLC-δ1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three Gi/o-coupled receptor agonists (somatostatin, δ-opioid agonist [D-Pen2,D-Pen5]enkephalin, and A1 agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-δ1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca2+ influx and was not observed in the absence of extracellular Ca2+, but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca2+ channels. Treatment of the cells with a Gq/13-coupled receptor agonist, CCK-8, caused only transient, PLC-β1-mediated PI hydrolysis. Unlike Gi/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-δ1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or Gα13 minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-δ1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca2+ influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine (∼25%) and strongly inhibited by SKF-96365 (∼75%) and in cells expressing PLC-δ1(E341R/D343R). Agonist-independent Ca2+ release or Ca2+ influx via voltage-gated Ca2+ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-δ1 antibody or nifedipine. We conclude that PLC-δ1 is activated by Gi/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca2+ influx via store-operated Ca2+ channels.

Novel bimodal effects of the G-protein tissue transglutaminase on adrenoreceptor signalling

Tissue transglutaminase (tTG) is a novel G-protein that previous studies showed can couple ligand-bound activated α1B adrenoreceptors to phospholipase C-δ, resulting in phosphoinositide (PI) hydrolysis. In human neuroblastoma SH-SY5Y cells we found that although endogenous tTG can facilitate α1B adrenoreceptor-stimulated PI hydrolysis, its contribution is minor compared with the classical heterotrimeric G-protein Gq/11. Further, we show that the α1B adrenoreceptor recruits tTG to the membrane and that this recruitment is enhanced by agonist occupancy of the receptor. In addition, the effects of tTG on signalling are bimodal. At low expression levels, tTG enhanced α1B adrenoreceptor-stimulated PI hydrolysis, whereas at higher expression levels tTG attenuated significantly this response. These findings are the first to demonstrate that a protein can both facilitate and attenuate receptor-stimulated PI hydrolysis.