Expression of mRNA encoding G protein-coupled receptors involved in congestive heart failure

Congestive heart failure (CHF) induces changes in the neurohumoral system and gene expression in viable myocardium. Several of these genes encode G protein-coupled receptors (GPCRs) involved in mechanisms which compensate for impaired myocardial function. We used real-time quantitative RT-PCR (Q-RT-PCR) to investigate the expression of mRNA encoding 15 different GPCRs possibly involved in CHF, and the effect of normalisation to GAPDH mRNA (GAPDH) or 18S rRNA (18S). CHF was induced in rats by coronary artery ligation, with sham-operated controls (Sham). After 6 weeks, mRNA expression in viable left ventricular myocardium was determined using both 18S and GAPDH as the normalisation standard. An apparent 30% reduction in GAPDH mRNA levels vs. 18S in CHF compared to Sham, although not significant in itself, influenced the interpretation of regulation of other genes.Thus, levels of mRNA encoding receptors for angiotensin II (AT₁), endothelin (ET_A, ET_B) and the muscarinic acetylcholine (mACh) receptor M₁ increased significantly in CHF only when normalised to GAPDH. Levels of mRNA encoding the mACh receptors M₃ and M₄ and the serotonin receptors 5-HT_2A and 5-HT₄ increased, whereas α_1D-adrenoceptor mRNA decreased in CHF irrespective of the normalisation standard. No significant change was detected for M2 and M5 mACh receptors or α_1A-, α_1B-, β₁- or β₂-adrenoceptors. Q-RT-PCR is a sensitive and powerful method to monitor changes in GPCR mRNA expression in CHF. However, the normalisation standard used is important for the interpretation of mRNA regulation. This article is accompanied by the Invited Editorial "Pitfalls in the normalization of real-time polymerase chain reaction data" by M. C. Hendriks-Balk et al. which can be found under     

Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca²⁺. This excessive Ca²⁺ liberation induces Ca²⁺-dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca²⁺ release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca²⁺ concentration in the thapsigargin-sensitive endoplasmic reticulum (ER) as well as in a bafilomycin-sensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca²⁺ release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca²⁺ release from the acid pool, that was mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP₃Rs) of types 2 and 3. POAEE evoked very little Ca²⁺ release and trypsin activation when IP₃Rs of both types 2 and 3 were knocked out. Antibodies against IP₃Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca²⁺ release and trypsin activation. We conclude that Ca²⁺ release through IP₃Rs of types 2 and 3 in the acid granular Ca²⁺ store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis.     

Reciprocal regulation of two G protein-coupled receptors sensing extracellular concentrations of Ca2+ and H+

G protein-coupled receptors (GPCRs) are cell surface receptors that detect a wide range of extracellular messengers and convey this information to the inside of cells. Extracellular calcium-sensing receptor (CaSR) and ovarian cancer gene receptor 1 (OGR1) are two GPCRs that sense extracellular Ca²⁺ and H⁺, respectively. These two ions are key components of the interstitial fluid, and their concentrations change in an activity-dependent manner. Importantly, the interstitial fluid forms part of the microenvironment that influences cell function in health and disease; however, the exact mechanisms through which changes in the microenvironment influence cell function remain largely unknown. We show that CaSR and OGR1 reciprocally inhibit signaling through each other in central neurons, and that this is lost in their transformed counterparts. Furthermore, strong intracellular acidification impairs CaSR function, but potentiates OGR1 function. Thus, CaSR and OGR1 activities can be regulated in a seesaw manner, whereby conditions promoting signaling through one receptor simultaneously inhibit signaling through the other receptor, potentiating the difference in their relative signaling activity. Our results provide insight into how small but consistent changes in the ionic microenvironment of cells can significantly alter the balance between two signaling pathways, which may contribute to disease progression.Cells are surrounded by interstitial fluid, the composition of which is influenced by neighboring cells and which constitutes a key part of the microenvironment in which cells have to operate and survive. Changes in this microenvironment influence cell physiology (1, 2) and may promote disease (3, 4). Extracellular Ca²⁺ ([Ca²⁺]_o) and H⁺ ([H⁺]_o) concentrations are important components of the microenvironment, and their extracellular concentration changes in an activity- and state-dependent manner (5, 6). [Ca²⁺]_o is required for membrane stability, serves as a reservoir to allow Ca²⁺ influx into cells, and contributes to the membrane potential. [H⁺]_o sets the local pH, thereby influencing protein function as well as contributing to the membrane potential. Furthermore, Ca²⁺ and H⁺ can cross the membrane via ion channels and transporters, meaning that both can serve as intracellular and extracellular messengers (7–10).Levels of [Ca²⁺]_o and [H⁺]_o are communicated to cells via cell surface receptors that change their activity in a manner dependent on [Ca²⁺]_o and [H⁺]_o. These receptors include G protein-coupled receptors (GPCRs), such as the extracellular Ca²⁺-sensing receptor (CaSR), ovarian cancer gene receptor 1 (OGR1), G protein-coupled receptor 4 (GPR4), and T-cell death-associated gene 8 (TDAG8), all of which sense [H⁺]_o, as well as a range of ion channels (8).Intriguingly, Ca²⁺ and H⁺ signaling can be intimately linked, and changes in extracellular pH (pH_o) and intracellular pH (pH_i) may affect intracellular Ca²⁺ ([Ca²⁺]_i) signaling directly and indirectly (8, 11, 12). This is exemplified by OGR1, which, like CaSR (13), can couple to G_q and hence trigger Ca²⁺ release from intracellular Ca²⁺ stores via activation of the phospholipase C pathway (14). Neither CaSR nor OGR1 desensitizes (13, 14); thus, they continually monitor [Ca²⁺]_o and [H⁺]_o levels, respectively, and faithfully report any changes in their extracellular concentration. Because of the vital importance of Ca²⁺ and H⁺ to cells, information about their extracellular presence is crucial for cells, and lack of or altered signaling through these receptors may contribute to disease pathways.We have previously found that OGR1 activation in DAOY cells, a human cerebellar granule cancer cell line, leads to complex [Ca²⁺]_i signals and activation of the ERK signaling pathway, thereby providing a mechanistic explanation of how the acidic environment may influence transformed cell function and/or survival (15). This action is lost on differentiation, suggesting a link between OGR1 activity and proliferative behavior of the transformed neurons (16). To better understand the role played by OGR1 in central neurons, we investigated OGR1 activation in primary cerebellar granule cells, the nontransformed equivalent of DAOY cells. We found that OGR1 and CaSR reciprocally inhibit [Ca²⁺]_i signaling through each other, and that intracellular acidosis, which accompanies extracellular acidification, promotes OGR1 but inhibits CaSR activity. Finally, CaSR-dependent inhibition of OGR1activity is absent in DAOY cells.     

Evidence for a Ca(2+)-gated ryanodine-sensitive Ca2+ release channel in visceral smooth muscle.

Although a role for the ryanodine receptor (RyR) in Ca2+ signaling in smooth muscle has been inferred, direct information on the biochemical and functional properties of the receptor has been largely lacking. Studies were thus carried out to purify and characterize the RyR in stomach smooth muscle cells from the toad Bufo marinus. Intracellular Ca2+ measurements with the Ca(2+)-sensitive fluorescent indicator fura-2 under voltage clamp indicated the presence of a caffeine- and ryanodine-sensitive internal store for Ca2+ in these cells. The (CHAPS)-solubilized, [3H]ryanodine-labeled RyR of toad smooth muscle was partially purified from microsomal membranes by rate density centrifugation as a 30-S protein complex. SDS/PAGE indicated the comigration of a high molecular weight polypeptide with the peak attributed to 30-S RyR, which had a mobility similar to the cardiac RyR and on immunoblots cross-reacted with a monoclonal antibody to the canine cardiac RyR. Following planar lipid bilayer reconstitution of 30-S stomach muscle RyR fractions, single-channel currents (830 pS with 250 mM K+ as the permeant ion) were observed that were activated by Ca2+ and modified by ryanodine. In vesicle-45Ca2+ efflux measurements, the toad channel was activated to a greater extent at 100-1000 microM than 1-10 microM Ca2+. These results suggest that toad stomach muscle contains a ryanodine-sensitive Ca2+ release channel with properties similar but not identical to those of the mammalian skeletal and cardiac Ca(2+)-release channels.     

Membrane repolarization stops caffeine-induced Ca2+ release in skeletal muscle cells.

We have combined the patch-clamp technique with fura-2 measurements to investigate whether the Ca(2+)-induced Ca(2+)-release channel is under the control of membrane potential in rat skeletal myoballs. We report that Ca2+ release induced by 10 mM caffeine is turned off by membrane repolarization, a phenomenon that we term RISC (repolarization-induced stop of Ca2+ release). The RISC phenomenon is voltage- and time-dependent. It is evident only when the release channels are first transferred into a functionally "voltage-activated" state through membrane depolarization. The results demonstrate that membrane repolarization actively closes the caffeine-activated release channels and suggest that the ryanodine receptor is actually the physiological depolarization-induced Ca(2+)-release channel. Thus, our data provide compelling evidence for a bidirectional voltage control (depolarization and repolarization) of the Ca(2+)-release channel in the sarcoplasmic reticulum by a voltage sensor in the transverse tubule membrane.     

Cyclic ADP-ribose contributes to contraction and Ca2+ release by M1 muscarinic receptor activation in coronary arterial smooth muscle

The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca(2+) release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 micromol/L) and oxotremorine (OXO; 2.5-80 micromol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca(2+) ([Ca(2+)](i)) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca(2+)](i) in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca(2+)-free Hanks' solution. This OXO-induced rise in [Ca(2+)](i) was significantly reduced by pirenzepine (PIR; an M(1) receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from beta-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca(2+) release induced by activation of M(1) muscarinic receptors.     

Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells

Aims/hypothesis

There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells.

Methods

We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents, intracellular Ca²⁺ ([Ca²⁺]_i) and insulin secretion in human beta cells.

Results

Extracellular application of ATP evoked small inward currents (3.4?±?0.7 pA) accompanied by depolarisation of the membrane potential (by 14.4?±?2.4 mV) and stimulation of electrical activity at 6 mmol/l glucose. ATP increased [Ca²⁺]_i by stimulating Ca²⁺ influx and evoking Ca²⁺ release via InsP₃-receptors in the endoplasmic reticulum (ER). ATP-evoked Ca²⁺ release was sufficient to trigger exocytosis in cells voltage-clamped at ?70 mV. All effects of ATP were mimicked by the P2Y_(1/12/13) agonist ADP and the P2Y₁ agonist MRS-2365, whereas the P2X_(1/3) agonist α,β-methyleneadenosine-5-triphosphate only had a small effect. The P2Y₁ antagonists MRS-2279 and MRS-2500 hyperpolarised glucose-stimulated beta cells and lowered [Ca²⁺]_i in the absence of exogenously added ATP and inhibited glucose-induced insulin secretion by 35%. In voltage-clamped cells subjected to action potential-like stimulation, MRS-2279 decreased [Ca²⁺]_i and exocytosis without affecting Ca²⁺ influx.

Conclusions/interpretation

These data demonstrate that ATP acts as a positive autocrine signal in human beta cells by activating P2Y₁ receptors, stimulating electrical activity and coupling Ca²⁺ influx to Ca²⁺ release from ER stores.     

Promoting effects of IL-13 on Ca2+ release and store-operated Ca2+ entry in airway smooth muscle cells

Th2 cytokine interleukin (IL)-13 plays a central role in the pathogenesis of allergic asthma. IL-13 exhibits a direct effect on airway smooth muscle cells (ASMCs) to cause airway hyperresponsiveness. IL-13 has been demonstrated to regulate Ca²⁺ signaling in ASMCs, but the underlying mechanisms are not fully understood. Store-operated Ca²⁺ entry (SOCE) plays an important role in regulating Ca²⁺ signaling and cellular responses of ASMCs, whether IL-13 affects SOCE in ASMCs has not been reported. In this study, by using confocal Ca²⁺ fluorescence imaging, we found that IL-13 (10 ng/ml) treatment increased basal intracellular Ca²⁺ ([Ca²⁺]_i) level, Ca²⁺ release and SOCE induced by SERCA inhibitor thapsigargin in rat bronchial smooth muscle cells. The glucocorticoid dexamethasone and the short-acting β2 adrenergic agonist (β2 agonist) salbutamol suppressed IL-13-augumented basal [Ca²⁺]_i, Ca²⁺ release and SOCE, whereas the long-acting β2 agonist salmeterol had no effect on altered Ca²⁺ signaling in IL-13-treated ASMCs. Membrane-permeable cAMP analog dibutyryl-cAMP (db-cAMP) similarly decreased Ca²⁺ release and SOCE induced by thapsigargin in IL-13-treated ASMCs, confirmed a role of cAMP/PKA signaling pathway in the regulation of SOCE. IL-13 promoted the proliferation of ASMCs stimulated by serum; this effect was inhibited by nonspecific Ca²⁺ channel blockers SKF-96365 and NiCl₂, by salmeterol, but not by salbutamol and dexamethasone. IL-13 treatment did not change the expression of SOC channel-associated molecules STIM1, Orai1 and TRPC1 at mRNA level. Our findings identified a promoting effect of IL-13 on Ca²⁺ release and SOCE in ASMCs, which partially contributes to its effect on the proliferation of ASMCs; the differences of glucocorticoids and β2 agonists in inhibiting Ca²⁺ signal and proliferation potentiated by IL-13 suggest that these therapies of asthma may have distinct effect on the relief of airway contraction and remodeling in bronchial asthma.     

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.     

Roles of G protein-coupled receptors in inflammatory bowel disease

Inflammatory bowel disease(IBD) is a complex disease with multiple pathogenic factors. Although the pathogenesis of IBD is still unclear, a current hypothesis suggests that genetic susceptibility, environmental factors, a dysfunctional immune system, the microbiome, and the interactions of these factors substantially contribute to the occurrence and development of IBD. Although existing and emerging drugs have been proven to be effective in treating IBD,none can cure IBD permanently. G protein-coupled receptors(GPCRs) are critical signaling molecules implicated in the immune response, cell proliferation,inflammation regulation and intestinal barrier maintenance. Breakthroughs in the understanding of the structures and functions of GPCRs have provided a driving force for exploring the roles of GPCRs in the pathogenesis of diseases, thereby leading to the development of GPCR-targeted medication. To date, a number of GPCRs have been shown to be associated with IBD, significantly advancing the drug discovery process for IBD. The associations between GPCRs and disease activity, disease severity, and disease phenotypes have also paved new avenues for the precise management of patients with IBD. In this review, we mainly focus on the roles of the most studied proton-sensing GPCRs, cannabinoid receptors,and estrogen-related GPCRs in the pathogenesis of IBD and their potential clinical values in IBD and some other diseases.     

Overexpression of G protein-coupled receptor kinase-2 in smooth muscle cells reduces neointimal hyperplasia

The activation of vascular smooth muscle cells (SMCs) in neointimal hyperplasia involves signaling through receptor tyrosine kinases as well as G protein-coupled receptors. Overexpression of G protein-coupled receptor kinase-2 (GRK2) in SMCs can attenuate mitogenic signaling and proliferation in response to not only several G protein-coupled receptor agonists, but also platelet-derived growth factor (PDGF). To test whether overexpression of GRK2 could inhibit other SMC responses implicated in neointimal hyperplasia, we assessed SMC chemotaxis and mitogenic signaling evoked by PDGF and G(q)-coupled receptor agonists. To test the effects of GRK2 overexpression on neointimal hyperplasia in vivo, we employed a rabbit autologous vein graft model system. GRK2 overexpression reduced PDGF-promoted SMC chemotaxis by 85% (P<0.01), but had no effect on chemotaxis promoted by epidermal growth factor (EGF). Congruently, GRK2 overexpression reduced by approximately 50% (P<0.05) the [(3)H]thymidine incorporation induced by combinations of PDGF and Gq-coupled receptor agonists, but had no effect on that induced by PDGF plus EGF. PDGF-, but not EGF-promoted phosphoinositide 3-kinase activity in SMCs was also inhibited by GRK2 overexpression. In rabbit vein grafts, we achieved GRK2 overexpression in medial SMCs, reduced cell proliferation during the first week after graft implantation, and reduced steady state neointimal thickness by 29% (P<0.01), without affecting medial thickness or potentiating SMC apoptosis. Because of its ability to dampen chemotactic and mitogenic signaling through PDGF and Gq-coupled receptors, GRK2 overexpression in SMCs may be a useful therapeutic approach for neointimal hyperplasia.     

Membrane hyperpolarization is not required for sustained muscarinic agonist-induced increases in intracellular Ca2+ in arteriolar endothelial cells

OBJECTIVE: Hyperpolarization modulates Ca2+ influx during agonist stimulation in many endothelial cells, but the effects of hyperpolarization on Ca2+ influx in freshly isolated arteriolar endothelial cells are unknown. Therefore, the purpose of the present study was to characterize agonist-induced Ca2+ transients in freshly isolated arteriolar endothelial cells and to test the hypothesis that membrane hyperpolarization augments agonist-induced Ca2+ influx into these cells. METHODS: Arterioles were removed from hamster cremaster muscles and arteriolar endothelial cells were enzymatically isolated. Endothelial cells were loaded with Fura 2-AM and the Fura 2 ratio measured photometrically as an index of intracellular Ca2+. The cells were then stimulated with the muscarinic, cholinergic agonist, methacholine, and the resulting Ca2+ transients were measured. RESULTS: Methacholine (1 microM) increased the endothelial cell Fura 2 ratio from a baseline of 0.81 +/- 0.02 to an initial peak of 1.17 +/- 0.05 (n = 17) followed by a sustained plateau of 1.12 +/- 0.07. The plateau phase of the Ca2+ transient was inhibited by removal of extracellular Ca2+ (n = 12, p < .05), or the nonselective cation channel blockers Gd3+ (30 microM; n = 7, p < .05) or La3+ (50 microM; n = 7, p < .05) without significant effect on the baseline or peak (p > .05). The initial peak of methacholine-induced Ca2+ transients was inhibited by the IP3-receptor antagonist xestospongin D (10 microM, n = 5, p < .05). The methacholine-induced Ca2+ transients were accompanied by endothelial cell hyperpolarization of approximately 14-18 mV, as assessed by experiments using the potentiometric dye, di-8-ANEPPS as well as by patch-clamp experiments. However, inhibition of hyperpolarization by blockade of Ca2+-activated K+ channels with charybdotoxin (100 nM) and apamin (100 nM) (n = 5), or exposure of endothelial cells to 80 or 145 mM KCl (both n = 7) had no effect on the plateau phase of methacholine-induced Ca2+ transients (p > .05). CONCLUSIONS: Freshly isolated arteriolar endothelial cells display agonist-induced Ca2+ transients. For the muscarinic agonist, methacholine, these Ca2+ transients result from release of Ca2+ from intracellular stores through IP3 receptors, followed by sustained influx of extracellular Ca2+. While these changes in intracellular Ca2+ are associated with endothelial cell hyperpolarization, the methacholine-induced, sustained increase in intracellular Ca2+ appears to be independent from this change in membrane potential. These data suggest that arteriolar endothelial cells may possess a novel Ca2+ influx pathway, or that the relationship between intracellular Ca2+ and Ca2+ influx is more complex than that observed in other endothelial cells.     

Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini

BACKGROUND & AIMS: The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) and the ryanodine receptor (RyR) are the principal Ca2+-release channels in cells and are believed to serve distinct roles in cytosolic Ca2+ (Ca(i)2+) signaling. This study investigated whether these receptors instead can release Ca2+ in a coordinated fashion. METHODS: Apical and basolateral Ca(i)2+ signals were monitored in rat pancreatic acinar cells by time-lapse confocal microscopy. Caged forms of second messengers were microinjected into individual cells and then photoreleased in a controlled fashion by either UV or 2-photon flash photolysis. RESULTS: InsP3 increased Ca(i)2+ primarily in the apical region of pancreatic acinar cells, whereas the RyR agonist cyclic adenosine diphosphate ribose (cADPR) increased Ca(i)2+ primarily in the basolateral region. Apical-to-basal Ca(i)2+ waves were induced by acetylcholine and initiation of these waves was blocked by the InsP3R inhibitor heparin, whereas propagation into the basolateral region was inhibited by the cADPR inhibitor 8-amino-cADPR. To examine integration of apical and basolateral Ca(i)2+ signals, Ca2+ was selectively released either apically or basolaterally using 2-photon flash photolysis. Ca(i)2+ increases were transient and localized in unstimulated cells. More complex Ca(i)2+ signaling patterns, including polarized Ca(i)2+ waves, were observed when Ca2+ was photoreleased in cells stimulated with subthreshold concentrations of acetylcholine. CONCLUSIONS: Polarized Ca(i)2+ waves are induced in acinar cells by serial activation of apical InsP3Rs and then basolateral RyRs, and subcellular release of Ca2+ coordinates the actions of these 2 types of Ca2+ channels. This subcellular integration of Ca2+-release channels shows a new level of complexity in the formation of Ca(i)2+ waves.     

Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.     

Aging impairs Ca2+ sensitization pathways in gallbladder smooth muscle

Calcium sensitization is an important physiological process in agonist-induced contraction of smooth muscle. In brief, calcium sensitization is a pathway that leads to smooth muscle contraction independently of changes in [Ca²⁺]_i by mean of inhibition of myosin light chain phosphatase. Aging has negative impacts on gallbladder contractile response due to partial impairment in calcium signaling and alterations in the contractile machinery. However, information regarding aging-induced alterations in calcium sensitization is scanty. We hypothesized that the calcium sensitization system is negatively affected by age. To investigate this, gallbladders were collected from adult (4 months old) and aged (22–24 months old) guinea pigs. To evaluate the contribution of calcium sensitization pathways we assayed the effect of the specific inhibitors Y-27632 and GF109203X on the “in vitro” isometric gallbladder contractions induced by agonist challenges. In addition, expression and phosphorylation (as activation index) of proteins participating in the calcium sensitization pathways were quantified by Western blotting. Aging reduced bethanechol- and cholecystokinin-evoked contractions, an effect associated with a reduction in MLC20 phosphorylation and in the effects of both Y-27632 and GF109203X. In addition, there was a drop in ROCK I, ROCK II, MYPT-1 and PKC expression and in the activation/phosphorylation of MYPT-1, PKC and CPI-17 in response to agonists. Interestingly, melatonin treatment for 4 weeks restored gallbladder contractile responses due to re-establishment of calcium sensitization pathways. These results demonstrate that age-related gallbladder hypocontractility is associated to alterations of calcium sensitization pathways and that melatonin treatment exerts beneficial effects in the recovery of gallbladder contractility.     

Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism

Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood.G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins which play essential roles in converting extracellular stimuli into intracellular signals in a variety of cell types. Odor detection by olfactory sensory neurons (OSNs) in the mammalian nose depends on a large family of G protein-coupled odorant receptors (ORs) (1), which endows the olfactory system with an extraordinary power of odor detection and discrimination. Although OR-ligand binding is the first step toward smell perception, little is known about the molecular and structural basis underlying odor response properties of individual ORs.Most mammalian ORs respond to a small fraction of all of the tested odorants (2). In contrast, recent studies have identified a small number of ORs that respond to a large set of diverse odorants with comparable potency and efficacy as the former. Curiously, several broadly responsive ORs including MOR256-3 (Olfr124 or SR1), MOR256-31 (Olfr263), and human OR2W1 (ortholog of MOR256-31) belong to the same subfamily, which also contains ORs such as MOR256-8 (Olfr1362) and MOR256-22 (Olfr1387) that respond to a few odorants (3–6). Identification of ORs within the same subfamily (i.e., sharing >50% amino acid identity) but with different response properties offers an opportunity for dissecting out the molecular features that define the tuning properties of these ORs.Mammalian ORs belong to class A (or rhodopsin family) GPCRs. The structure-function relationship of several class A members (e.g., rhodopsin and β2-adrenergic receptor) has been investigated in great details via various approaches including site-directed mutagenesis, X-ray crystallography (7, 8), and molecular modeling (9–11). Although no crystal structure is available for any OR, site-directed mutagenesis and/or computational modeling have shed light on structure-function relationship for a few ORs (12–18).Using a joint approach of site-directed mutagenesis and computational modeling, we investigated the response properties of mutant ORs based on MOR256-3 and MOR256-8, which responded to a large and small set of odorants, respectively. Three-dimensional atomic models of these ORs were built to map locations of the mutated residues. Most mutations in MOR256-3 altered the responses to different odorants in a similar manner. Remarkably, MOR256-8 was converted into a broadly responsive OR by swapping a single or a few residues. More generally, we found that an OR’s total response was positively correlated with its basal activity, an indication of ligand-independent receptor activation. These data suggest that broad responsiveness of an OR is not only determined by ligand binding, but also by activation mechanism.