Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca²⁺]_i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCδ1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca²⁺]_i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET_AR) antagonist, BQ123. To characterize ET_AR desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET_AR responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET_AR desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive ^D110A,K220RGRK2, ^D110A,K220RGRK3, ^K215RGRK5, or ^K215RGRK6 constructs. ^D110A,K220RGRK2 expression significantly attenuated ET_AR desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET_AR desensitization. Finally, immunocyotchemical data showed that ET_AR activation recruited endogenous GRK2 from cytoplasm to membrane. These studies identify GRK2 as a key regulator of ET_AR responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.

We have previously shown that overexpression of G protein-coupled receptor kinase 6 (GRK6) enhanced the phosphorylation and desensitization of the endogenously expressed M₃muscarinic acetylcholine (mACh) receptor in human SH-SY5Y neuroblastoma cells. In this study we have examined the potential role of endogenous GRK6 in the regulation of M₃ mACh receptor by blocking its action through the introduction of a kinase-dead, dominant-negative GRK6 (^K215RGRK6). ^K215RGRK6 expression inhibited methacholine-stimulated M₃ mACh receptor phosphorylation by 50% compared with plasmid transfected control cells. Guanosine-5′-O-(3-[³⁵S]thio)triphosphate binding and immunoprecipitation studies, conducted after agonist pretreatment (3 min), indicated that M₃ mACh receptor-Gα_q/11 uncoupling was attenuated by 50% in cells expressing ^K215RGRK6 when compared with control cells. In contrast, expression of the related dominant-negative kinase^K215RGRK5 had no effect on M₃ mACh receptor phosphorylation or uncoupling. Time course studies also showed that agonist-stimulated [³H]inositol phosphate accumulations were more sustained in cells expressing ^K215RGRK6 compared with control and ^K215RGRK5-expressing cells, whereas^K215RGRK6 expression had no effect on the phospholipase C response to direct stimulation of G proteins with AlF . The ability of^K215RGRK6 to inhibit agonist-mediated M₃ mACh receptor phosphorylation and G protein uncoupling suggests that endogenous GRK6 mediates, at least in part, M₃ mACh receptor desensitization in the SH-SY5Y cell line.

BACKGROUND AND PURPOSE: The uterotonins oxytocin and histamine, mediate contractile signals through specific G protein‐coupled receptors, a process which is tightly controlled during gestation to prevent preterm labour. We previously identified G protein‐coupled receptor kinase (GRK)2 and GRK6 as respective cardinal negative regulators of histamine H₁ and oxytocin receptor signalling. GRK‐mediated phosphorylation promotes arrestin recruitment, not only desensitizing receptors but activating an increasing number of diverse signalling pathways. Here we investigate potential roles that arrestins play in the regulation of myometrial oxytocin/histamine H₁ receptor signalling.EXPERIMENTAL APPROACH: Endogenous arrestins2 and 3 were specifically depleted using RNA‐interference in a human myometrial cell line and the consequences of this for G protein‐coupled receptor‐mediated signalling were assessed using Ca²⁺/inositol 1,4,5‐trisphophate imaging and standard mitogen‐activated protein kinase (MAPK) assays.KEY RESULTS: Depletion of arrestin3, but not arrestin2 enhanced and prolonged H₁ receptor‐stimulated Ca²⁺ responses, whilst depletion of either arrestin increased oxytocin receptor responses. Arrestin3 depletion decreased H₁ receptor desensitization, whilst removal of either arrestin isoform was equally effective in preventing oxytocin receptor desensitization. Following arrestin3 depletion oxytocin‐induced phospho‐extracellular signal‐regulated kinase1/2 signals were diminished and histamine‐stimulated signals virtually absent, whereas depletion of arrestin2 augmented extracellular signal‐regulated kinase1/2 responses to each agonist. Conversely, depletion of arrestin3 enhanced p38 signals to each agonist, whilst arrestin2 suppression increased oxytocin‐, but not histamine‐induced p38 MAPK responses.CONCLUSIONS AND IMPLICATIONS: Arrestin proteins are key regulators of H₁ and oxytocin receptor desensitization, and play integral roles mediating uterotonin‐stimulated MAPK‐signalling. These data provide insights into the in situ regulation of these receptor subtypes and may inform pathophysiological functioning in preterm labour.

Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [³H]inositol phosphate accumulation and intracellular Ca²⁺ concentration ([Ca²⁺]_i); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific ≥75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

We have investigated the effects of G protein-coupled receptor kinase (GRK) 3 and GRK6 on the phosphorylation and regulation of the M₃ muscarinic acetylcholine receptor (mACh) endogenously expressed in SH-SY5Y cells. Overexpression of GRK3 or GRK6 enhanced M₃ mACh receptor phosphorylation after high-concentration methacholine (100 μM, 1 min) addition. However, GRK6 was more potent, increasing receptor phosphorylation even after low (3 μM, 1 min) agonist stimulation. Compared with plasmid-transfected control cells expressing equivalent M₃ mACh receptor number, GRK3- or GRK6-overexpressing cells exhibited a reduced phospholipase C activity reflected by a lower accumulation of total [³H]inositol phosphates and Ins(1,4,5)P₃ mass. In addition, direct stimulation of G protein activation of phospholipase C (by AlF₄⁻) was inhibited in GRK3- but not GRK6-overexpressing cells. Guanosine-5′-O-(3-[³⁵S]thio)triphosphate binding and immunoprecipitation of Gα_q/11 indicated that acute methacholine-stimulated receptor/Gα_q/11 coupling was unaffected by GRK overexpression. In contrast, agonist pretreatment of cells for 3 min caused M₃ mACh receptor uncoupling from Gα_q/11, which was markedly enhanced by GRK6 overexpression, particularly at lower agonist pretreatment concentrations. However, the increased M₃ mACh receptor phosphorylation seen in clones overexpressing GRK3 was not accompanied by increased receptor-Gα_q/11 uncoupling. Overall, these data suggest that GRK3 and GRK6 use different pathways to desensitize the M₃ mACh receptor. GRK6 seems to act as a classical GRK, inducing increased receptor phosphorylation accompanied by an uncoupling of receptor and Gα_q/11. Conversely, GRK3 may cause desensitization independently of receptor phosphorylation, possibly via Gβγ binding and/or direct Gα_q binding via its regulator of G protein signaling domain to inhibit phospholipase C activity.