EDITORIAL FOCUS
cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Karnam S. Murthy and Gabriel M. Makhlouf

Departments of Medicine and Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Recent studies on the role of nitric oxide (NO) in gastrointestinal smooth muscle have raised the possibility that NO-stimulated cGMP could, in the absence of cGMP-dependent protein kinase (PKG) activity, act as a Ca2+-mobilizing messenger [K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G660-G671, 1993]. This notion was examined in dispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) and with NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM), NO (1 µM), and VIP (1 µM) stimulated 45Ca2+ release (21 ± 3 to 30 ± 1% decrease in 45Ca2+ cell content); Ca2+ release stimulated by 8-BrcGMP was concentration dependent with an EC50 of 0.4 ± 0.1 µM and a threshold of 10 nM. 8-BrcGMP and NO increased cytosolic free Ca2+ concentration ([Ca2+]i) and induced contraction; both responses were abolished after Ca2+ stores were depleted with thapsigargin. With VIP, which normally increases [Ca2+]i by stimulating Ca2+ influx, treatment with PKA and PKG inhibitors caused a further increase in [Ca2+]i that reverted to control levels in cells pretreated with thapsigargin. Neither Ca2+ release nor contraction induced by cGMP and NO in permeabilized muscle cells was affected by heparin or ruthenium red. Ca2+ release induced by maximally effective concentrations of cGMP and inositol 1,4,5-trisphosphate (IP3) was additive, independent of which agent was applied first. We conclude that, in the absence of PKA and PKG activity, cGMP stimulates Ca2+ release from an IP3-insensitive store and that its effect is additive to that of IP3.

cytosolic free calcium; cAMP-dependent protein kinase; cGMP-dependent protein kinase; calcium stores

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THREE MAJOR TARGETS for cGMP have been identified: cGMP-dependent protein kinases (types Ialpha , Ibeta , and II) (22, 38, 42); cGMP-gated cation channels (6, 16); and various cyclic nucleotide phosphodiesterases (PDE), including cGMP-specific PDE5, cGMP-stimulated PDE2, and cGMP-inhibited PDE3 (2). cGMP, acting via cGMP-dependent protein kinase (PKG), has distinct effects on intracellular Ca2+ levels in different cells, increasing cytosolic free Ca2+ concentration ([Ca2+]i) in hepatocytes (36) and sea urchin eggs (12, 13, 20) and decreasing [Ca2+]i in vascular (8) and visceral smooth muscle (27, 28), cardiac myocytes (23), and cerebellar neurons (3). In hepatocytes, phosphorylation of the inositol 1,4,5-trisphosphate (IP3) receptor by PKG potentiates IP3-dependent Ca2+ release (36), whereas in sea urchin eggs phosphorylation of ADP ribosyl cyclase stimulates the synthesis of the Ca2+-mobilizing messenger, cyclic ADP-ribose, which potentiates Ca2+-induced Ca2+ release from ryanodine-sensitive stores (12, 13). In smooth muscle, on the other hand, phosphorylation of a different IP3 receptor isoform by PKG inhibits IP3-dependent Ca2+ release (17, 41). In addition, PKG acts on other targets in smooth muscle to attenuate [Ca2+]i. Thus PKG 1) inhibits the activity of phospholipase C-beta and generation of IP3 (28); 2) stimulates the activity of plasmalemmal (36, 44) and sarcoplasmic Ca2+-ATPase pumps (9, 34, 39), thereby increasing Ca2+ uptake into the stores and Ca2+ efflux from the cells; and 3) inhibits the activity of Ca2+ channels (23) and stimulates the activity of K+ channels in the plasma membranes, thereby reducing Ca2+ influx into the cells via voltage-sensitive Ca2+ channels (4).

The diverse and marked effects of PKG on Ca2+ mobilization in smooth muscle have hampered an assessment of a direct role for cGMP. cGMP can directly activate cationic channels of the outer segment of the retinal rod and in the cilia of olfactory receptor neurons (6, 30) and Ca2+ channels in pancreatic acinar cells and NIH/3T3 cells, leading to sustained Ca2+ influx (1, 31, 43). Our recent studies (25, 27) on the regulatory role of nitric oxide (NO) in gastric and intestinal smooth muscle cells have raised the possibility that cGMP could, in the absence of PKG activity, induce Ca2+ mobilization. The transient increase in [Ca2+]i that accompanies stimulation of NO and cGMP formation in muscle cells by vasoactive intestinal peptide (VIP) is enhanced when cAMP-dependent protein kinase (PKA) and PKG activity is blocked, converting the smooth muscle response from relaxation to contraction (29). We have postulated that a product of the cascade that results in activation of PKG could be responsible for the increase in [Ca2+]i. In the present study, we have examined the possibility that cGMP can act as a Ca2+mobilizing messenger in the absence of protein kinase activity. The effect of cGMP was tested with the permeant derivative of cGMP, 8-bromo-cGMP (8-BrcGMP), and with NO and VIP, which stimulate endogenous cGMP formation. The results indicate that cGMP stimulates Ca2+ release from an IP3-insensitive store and that its effect is additive to that of IP3.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Dispersion of smooth muscle cells. Muscle cells were isolated from the circular muscle layer of the rabbit stomach by successive enzymatic digestion, filtration, and centrifugation as described previously (25, 29). After digestion, the muscle cells were resuspended in enzyme-free medium consisting of (in mM) 120 NaCl, 4 KCl, 2.6 KH2PO4, 0.6 MgCl2, 25 HEPES, 14 glucose, and 2.1% Eagle's essential amino acid mixture. The cells were harvested by filtration through 500-µm Nitex mesh and centrifuged twice at 350 g for 10 min. In some experiments, the cells were permeabilized by incubation with saponin (35 µg/ml) for 10 min as previously described (18, 28). The cells were centrifuged at 350 g for 10 min, washed free of saponin, and resuspended in a medium containing 100 nM Ca2+ and an ATP-regenerating system consisting of 5 mM creatine phosphate and 10 U/ml creatine phosphokinase.

Measurement of [Ca2+]i in dispersed smooth muscle cells. [Ca2+]i was measured in suspensions of muscle cells using the Ca2+ fluorescent dye fura 2, as described previously (24, 29). Muscle cells were suspended in a medium containing (in mM) 10 HEPES, 125 NaCl, 5 KCl, 1 CaCl2, 0.5 MgSO4, 5 glucose, 20 taurine, 45 sodium pyruvate, and 5 creatine and were incubated with fura 2-AM (2 µM) for 20 min at 31°C. After centrifugation at 350 g for 20 min, the cells were incubated in fura 2-free medium for immediate measurement of Ca2+. Fluorescence was monitored at 510 nm, with excitation wavelengths alternating between 340 and 380 nm, and the measurements were corrected for autofluorescence of unloaded cells. An estimate of [Ca2+]i was obtained from observed, maximal, and minimal fluorescence ratios as described previously (24, 29).

Measurement of 45Ca2+ release in dispersed smooth muscle cells. Net Ca2+ efflux was measured in dispersed muscle cells as described previously (28, 32). The cells were incubated in a medium containing 45Ca2+ (10 µCi/ml) and antimycin (10 µM), and Ca2+ uptake into nonmitochondrial Ca2+ stores was measured at intervals for 60 min when a steady state was attained. VIP, 8-BrcGMP, or NO was then added, and net Ca2+ efflux was measured after 30 s. Net Ca2+ efflux, reflecting release from nonmitochondrial stores, was expressed as percent decrease in steady-state 45Ca2+ cell content. A similar procedure was followed in permeabilized muscle cells suspended in a medium containing 100 nM Ca2+ and antimycin (10 µM) to prevent mitochondrial Ca2+ uptake. ATP (1.5 mM) in the presence of an ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine phosphokinase) was first added to initiate Ca2+ uptake into the nonmitochondrial stores. After 60 min, VIP, 8-BrcGMP, or NO was added, and net Ca2+ efflux was measured after 30 s. Net Ca2+ efflux was expressed as percent decrease in steady-state 45Ca2+ cell content. Steady-state 45Ca2+ cell content in permeabilized muscle cells (2.63 ± 0.32 nmol/106 cells) was not significantly different from that in intact muscle cells (2.19 ± 0.26 nmol/106 cells).

Assay for NO synthase activity in dispersed smooth muscle cells. NO synthase (NOS) activity was measured in dispersed muscle cells as described previously (15, 25, 29) from the stoichiometric formation of the coproduct, L-citrulline in cells preloaded with L-[3H]arginine. L-[3H]arginine (3 µCi/ml) was added to 1 ml of cell suspension for 10 min, and the cells were treated during the last minute with either VIP, 8-BrcGMP, or NO. The suspension was centrifuged for 1 min at 3,000 g and the pellet was frozen rapidly on dry ice. The samples were stored at -70°C for extraction and separation of L-[3H]citrulline by flow-through chromatography using Dowex (AG50W-X8) columns. The amount of L-[3H]citrulline in the sample was measured by liquid scintillation. The results were expressed as counts per minute (cpm) per 106 cells or as percent increase above basal levels measured in separate aliquots of the same cell suspension. Previous studies had shown that this assay yielded results similar to measurement of the end products, NO-3/NO-2, by HPLC (15, 25).

Measurement of cell contraction. Contraction of dispersed muscle cells was measured by scanning micrometry as described previously (18, 24). Briefly, an aliquot (0.5 ml) of cell suspension containing 104 muscle cells/ml was added to 0.2 ml of HEPES medium containing VIP, NO, or 8-BrcGMP for 30 s. The reaction was terminated by addition of acrolein to a final concentration of 1%. The length of muscle cells treated with each agent was measured by scanning micrometry and was compared with the length of untreated cells (mean control cell length: 101 ± 5 µm). Contraction was expressed as the decrease in micrometers from mean control cell length.

Materials. 125I-labeled cGMP, L-[3H]arginine, and 45Ca2+ were obtained from NEN Life Sciences Products; HEPES was from Research Organics (Cleveland, OH); soybean trypsin inhibitor and collagenase (type II) were from Worthington; fura 2-AM was from Molecular Probes; thapsigargin, ruthenium red, 6-anilino-5,8-quinolinedione (LY-83583), and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide hydrochloride (H-89) were from Calbiochem; (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H,-2,7b,11a-trizadibenzo(a,g)cycloocta(cde)-trinden-1-one (KT-5823) was from Kamiya Biomedical (Thousand Oaks, CA); low-molecular-weight heparin, NG-nitro-L-arginine (L-NNA) and all other chemicals were from Sigma Chemical.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Ca2+ release and contraction induced by cGMP in dispersed smooth muscle cells. The ability of 8-BrcGMP to stimulate Ca2+ release from sarcoplasmic stores was examined in muscle cells loaded with 45Ca2+ and treated for 10 min with the PKA inhibitor H-89 (1 µM) and the PKG inhibitor KT-5823 (1 µM). These concentrations were previously shown to be both selective and maximally effective in inhibiting PKA and PKG, respectively, based on direct measurement of PKA and PKG activity in these smooth muscle cells (27). In other tissues, H-89 at a concentration of 1 µM was shown also to inhibit PKG (7, 37). Both protein kinase inhibitors were used so as to preclude effects that might result from cross-activation of PKA. The use of both PKA and PKG inhibitors was especially important in experiments with VIP, which stimulates both cAMP and cGMP and activates both protein kinases (27, 28).

In the presence of both protein kinase inhibitors, 8-BrcGMP caused a concentration-dependent increase in Ca2+ release (Fig. 1). Maximal release (30 ± 1% decrease in steady-state 45Ca2+ cell content; P < 0.001, n = 4) was similar to that elicited by a maximally effective concentration of IP3 (1 µM) in permeabilized muscle cells (32 ± 3% decrease in steady-state 45Ca2+ cell content; P < 0.01, n = 4) (18, 24, 27). The EC50 was 0.4 ± 0.1 µM with a threshold concentration of ~10 nM. No Ca2+ release was observed in the absence of PKA and PKG inhibitors (Fig. 1).


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Fig. 1.   Stimulation of Ca2+ release by 8-bromo-cGMP (8-BrcGMP) in dispersed gastric smooth muscle cells. Muscle cells were incubated in a medium containing 45Ca2+ (10 µCi/ml) and antimycin (10 µM), and Ca2+ uptake into nonmitochondrial Ca2+ stores was measured at intervals for 60 min when a steady state was attained. 8-BrcGMP was then added, and net Ca2+ efflux was measured after 30 s in presence or absence of cAMP-dependent protein kinase (PKA) inhibitor, H-89 (1 µM), and cGMP-dependent protein kinase (PKG) inhibitor, KT-5823 (1 µM). Net Ca2+ efflux, reflecting Ca2+ release from nonmitochondrial stores, was expressed as percent decrease in steady-state 45Ca2+ cell content. Steady-state 45Ca2+ cell content in these experiments was 2.29 ± 0.28 nmol/106 cells. Values are means ± SE of 4 experiments.

Ca2+ release in the presence of PKA and PKG inhibitors was observed also with NO (21 ± 3% decrease in 45Ca2+ cell content; P < 0.01, n = 4) and VIP (23 ± 1%; P < 0.001, n = 4), which were used at maximally effective concentrations (Fig. 2) (15, 29). The effects of NO and VIP on Ca2+ release were attributed to stimulation of endogenous cGMP. The increase in cGMP induced by NO (0.52 ± 0.05 pmol/106 cells above a basal level of 0.45 ± 0.04 pmol/106 cells; P < 0.01, n = 4) was not affected by the presence of PKA and PKG inhibitors (0.49 ± 0.03 pmol/106 cells) or L-NNA (0.51 ± 0.05 pmol/106 cells), whereas the increase in cGMP induced by VIP (0.51 ± 0.01 pmol/106 cells; P < 0.001, n = 4) was abolished by L-NNA (0.03 ± 0.04 pmol/106 cells). Suppression of VIP-stimulated NO and cGMP formation by L-NNA abolished Ca2+ release induced by VIP, consistent with the absence of Ca2+-induced Ca2+ release in circular smooth muscle cells (Fig. 2) (18, 19). L-NNA had no effect on Ca2+ release induced by NO or 8-BrcGMP.


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Fig. 2.   Ca2+ release and contraction induced by 8-BrcGMP, nitric oxide (NO), and vasoactive intestinal peptide (VIP) in dispersed smooth muscle cells. Muscle cells were loaded with 45Ca2+ as described in legend to Fig. 1. 8-BrcGMP (10 µM), NO (1 µM), or VIP (1 µM) was then added, and net Ca2+ efflux was measured after 30 s in presence or absence of PKA and PKG inhibitors (1 µM each) and in presence of NO synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA; 100 µM). Cells were treated for 10 min with inhibitors before addition of 8-BrcGMP, NO, or VIP. Net Ca2+ efflux (A) was expressed as percent decrease in steady-state 45Ca2+ cell content (2.08 ± 0.23 nmol/106 cells in these experiments). Contraction (B) was measured by scanning micrometry and expressed as decrease in cell length from control (control cell length: 97 ± 3 µm). Values are means ± SE of 3-4 experiments. ** P < 0.01, inhibition of VIP-induced Ca2+ release and contraction by L-NNA.

Coincidentally with stimulation of Ca2+ release, all three agents (8-BrcGMP, NO, and VIP) induced muscle cell contraction (Fig. 2). The contraction induced by 8-BrcGMP (18 ± 2 µm decrease in cell length; P < 0.01, n = 4) or NO (19 ± 1 µm; P < 0.001, n = 4) was not affected by treatment of the cells with L-NNA (100 µM), whereas the contraction induced by VIP (21 ± 1 µm; P < 0.001, n = 4) was partly inhibited (10 ± 2 µm) (Fig. 2); the residual contraction was attributed to stimulation of Ca2+ influx by VIP. Previous studies (25) had shown that L-NNA had no effect on VIP-stimulated Ca2+ influx, a step proximal to that of NOS activation and cGMP formation.

[Ca2+]i and muscle contraction induced by 8-BrcGMP, NO, and VIP. As expected from measurements of 45Ca2+ release, both 8-BrcGMP and NO increased [Ca2+]i measured in fura 2-loaded cells (8-BrcGMP to 140 ± 55 nM, P < 0.05, and NO to 89 ± 6 nM, P < 0.01, respectively, above a basal level of 56 ± 2 nM Ca2+); the increase in [Ca2+]i was observed only after treatment of the cells with PKA and PKG inhibitors (Fig. 3). VIP increased [Ca2+]i (160 ± 20 nM above basal level; P < 0.01, n = 4) in the absence of protein kinase inhibitors; the increase was caused by Ca2+ influx and was abolished by nifedipine (11 ± 6 nM; not significant). The increase in VIP-induced [Ca2+]i was significantly augmented (P < 0.02) after treatment of the cells with PKA and PKG inhibitors (314 ± 50 nM; Fig. 3).


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Fig. 3.   Muscle cell contraction and increase in cytosolic free Ca2+ concentration ([Ca2+]i) induced by 8-BrcGMP, NO, and VIP in dispersed smooth muscle cells. [Ca2+]i (A) was measured in suspensions of smooth muscle cells loaded with fura 2 as described in METHODS and expressed as increase in [Ca2+]i above basal level (mean basal: 56 ± 2 nM). Contraction (B) was measured by scanning micrometry and expressed as decrease in cell length from control (105 ± 4 µm). [Ca2+]i and contraction in response to 8-BrcGMP (10 µM), NO (1 µM), or VIP (1 µM) were measured before and after treatment of cells with thapsigargin and with PKA and PKG inhibitors (1 µM each). Thapsigargin (2 µM) in zero Ca2+ plus EGTA was first added for 30 min followed by restoration of normal Ca2+ levels. Contraction and increase in [Ca2+]i in presence of protein kinase inhibitors were significant for all agents (P < 0.01). A significant increase in VIP-induced [Ca2+]i (P < 0.01) in absence of protein kinase inhibitors reflected Ca2+ influx. Values are means ± SE of 3-4 experiments. ** Inhibition of contraction and [Ca2+]i after treatment with thapsigargin; P < 0.01. 

After treatment with thapsigargin to deplete the Ca2+ stores (30-min incubation with 2 µM thapsigargin in zero Ca2+ plus EGTA followed by resuspension of the cells in control 2 mM Ca2+ medium), there was no increase in [Ca2+]i with 8-BrcGMP and NO in the presence of PKA and PKG inhibitors, whereas the increase in [Ca2+]i induced by VIP reverted to control levels reflecting Ca2+ influx (123 ± 16 nM above basal levels; Fig. 3).

The pattern of muscle cell contraction paralleled the changes in [Ca2+]i. In the presence of PKA and PKG inhibitors, all three agents induced muscle cell contraction (Fig. 3). The contraction induced by 8-BrcGMP (18 ± 2 µm decrease in cell length; P < 0.01, n = 4) and NO (19 ± 1 µm; P < 0.001, n = 4) was abolished after treatment of the cells with thapsigargin, whereas the contraction induced by VIP (21 ± 1 µm; P < 0.001, n = 4) was partly inhibited; the residual contraction (11 ± 2 µm) reflected the effect of Ca2+ influx (Fig. 3).

Effect of 8-BrcGMP, NO, and VIP on NOS activity. The changes in [Ca2+]i were reflected by corresponding changes in NOS activity of dispersed smooth muscle cells. In the absence of PKA and PKG inhibitors, neither NO (1 µM) nor 8-BrcGMP (10 µM) stimulated NOS activity, whereas VIP (1 µM) caused a significant increase in NOS activity (716 ± 114 cpm/106 cells above basal level of 1,010 ± 133 cpm; P < 0.01, n = 4), similar to that previously reported for VIP and shown to be dependent on VIP-stimulated Ca2+ influx (Fig. 4) (29). In the presence of PKA and PKG inhibitors, both NO and 8-BrcGMP caused significant increases in NOS activity (520 ± 49 and 297 ± 43 cpm/106 cells above basal level, respectively; P < 0.01, n = 4); the VIP-induced increase in NOS activity was not altered by treatment with protein kinase inhibitors (728 ± 116 cpm/106 cells).


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Fig. 4.   NOS activity stimulated by 8-BrcGMP, NO, and VIP in dispersed smooth muscle cells. NOS activity was measured as L-[3H]citrulline formation in cells loaded with L-[3H]arginine. 8-BrcGMP (10 µM), NO (1 µM), or VIP (1 µM) was added for 60 s, and responses were measured in presence or absence of PKA and PKG inhibitors (1 µM) or L-NNA (100 µM). Responses were also measured after 30-min treatment with thapsigargin (2 µM) to deplete Ca2+ stores (see legend to Fig. 3). Results are expressed as cpm/106 cells above basal level (basal: 1,010 ± 133 cpm/106 cells). Significant increase of NOS activity induced by 8-BrcGMP and NO coincided with increase in Ca2+ release (see Figs. 2 and 3). Values are means ± SE of 4 experiments. ** P < 0.01, inhibition of NOS activity by L-NNA and after treatment with thapsigargin.

Treatment of the cells with L-NNA (100 µM) abolished NOS activity induced by all three agents, whereas treatment with thapsigargin abolished only the NOS activity induced by NO and 8-BrcGMP (Fig. 4). The results implied that activation of NOS by 8-BrcGMP and NO was mediated by cGMP-dependent Ca2+ release from thapsigargin-sensitive Ca2+ stores. The resultant increase in [Ca2+]i activated a constitutive NOS in smooth muscle (26).

Identity of cGMP-sensitive Ca2+ stores. In permeabilized smooth muscle cells treated with PKA and PKG inhibitors, Ca2+ release induced by 10 µM cGMP (26 ± 2% decrease in 45Ca2+ steady-state cell content; P < 0.001, n = 4) or 1 µM NO (25 ± 2%; P < 0.001, n = 4) was not affected by 10 or 100 µg/ml heparin (range: 24 ± 2 to 27 ± 4%) or by 10 and 100 µM ruthenium red (range: 22 ± 2 to 26 ± 2%). The corresponding contractions induced by cGMP (22 ± 5 µm decrease in cell length; P < 0.02, n = 4) and NO (21 ± 4 µm; P < 0.01, n = 4) were also not affected by heparin (range: 21 ± 3 to 24 ± 4 µm) or ruthenium red (20 ± 4 to 24 ± 3 µm). In contrast, Ca2+ release (32 ± 3% decrease in 45Ca2+ cell content; P < 0.01, n = 4) and contraction (28 ± 5 µm decrease in cell length; P < 0.01, n = 4) induced by 1 µM IP3 were abolished by heparin (10 µg/ml). The lack of effect of ruthenium red or heparin on cGMP-stimulated Ca2+ release suggested that cGMP did not activate IP3 receptor/Ca2+ channels or ryanodine receptor/Ca2+ channels and may have acted on distinct, nonmitochondrial Ca2+ stores.

These notions were corroborated in studies using permeabilized muscle cells treated sequentially with cGMP and IP3. After loading with 45Ca2+ for 60 min, the cells were treated with protein kinase inhibitors and thapsigargin. Addition of a maximally effective concentration of cGMP (10 µM) caused a prompt release of Ca2+ (26 ± 3% decrease in 45Ca2+ cell content within 30 s; P < 0.01; n = 4); subsequent addition of IP3 in the continued presence of cGMP caused a further release of Ca2+ (32 ± 2% decrease in 45Ca2+ cell content within 15 s; P < 0.01, n = 4); the combined effect of cGMP and IP3 caused a 58 ± 3% decrease in 45Ca2+ steady-state cell content (Fig. 5). Similar additive effects were obtained when IP3 was added first, followed by cGMP, and were evident when a longer interval (10 min) separated the addition of the two agents (Fig. 5). Addition of ionomycin (10 µM) caused further Ca2+ release, virtually depleting the Ca2+ stores (87 ± 4%). Addition of GTP (10 µM) to permeabilized gastric smooth muscle cells did not elicit significant Ca2+ release (2.5% decrease in 45Ca2+ cell content).


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Fig. 5.   Effect of sequential addition of cGMP and inositol 1,4,5-trisphosphate (IP3) on Ca2+ release from permeabilized smooth muscle cells. Ca2+ release in muscle cells loaded with 45Ca2+ was measured as described in METHODS. After steady-state Ca2+ uptake was attained, either cGMP (10 µM) or IP3 (1µM) was added in presence of thapsigargin to prevent Ca2+ reuptake and in presence of PKA and PKG inhibitors (1 µM each). After a 2-min interval (A) or 10-min interval (B), the other agent was added. Ionomycin (IM; 10 µM) was added at end of experimental period. Net Ca2+ efflux was expressed as percent decrease in steady-state 45Ca2+ cell content (2.48 ± 0.25 nmol/106 cells). Values are means ± SE of 4 experiments.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Three agents (8-BrcGMP, NO, and VIP) that normally cause relaxation of smooth muscle could be converted to contractile agents in the presence of PKA and PKG inhibitors. The agents, which have in common the ability to increase intracellular levels of cGMP, stimulated Ca2+ release from IP3-insensitive sarcoplasmic Ca2+ stores, leading to an increase in [Ca2+]i and smooth muscle cell contraction.

The Ca2+ release induced by these agents was clearly attributable to cGMP. In the presence of protein kinase inhibitors, exogenous 8-BrcGMP stimulated Ca2+ release in a concentration-dependent fashion with an EC50 of 0.4 ± 0.1 µM and a threshold concentration of 10 nM. The resultant increase in [Ca2+]i induced by 8-BrcGMP (or by NO that stimulated endogenous cGMP) was abolished after depletion of the Ca2+ stores with thapsigargin. The increase in [Ca2+]i induced by VIP reflected VIP-stimulated Ca2+ influx (29) as well as cGMP-dependent Ca2+ release: only the latter component was eliminated after treatment of the muscle cells with thapsigargin; the residual increase in [Ca2+]i reflecting Ca2+ influx was not affected by protein kinase inhibitors but was abolished by the dihydropyridine Ca2+ channel blocker, nifedipine (25, 29). The possibility that VIP-induced Ca2+ influx could have led to Ca2+-induced Ca2+ release was ruled out by the fact that L-NNA, which suppresses VIP-stimulated NOS activity and cGMP formation but has no effect on VIP-stimulated Ca2+ influx, suppressed VIP-induced Ca2+ release (25, 29). It is worth noting that the increase in [Ca2+]i induced by 8-BrcGMP and NO in the presence of protein kinase inhibitors led to activation of the constitutive smooth muscle NOS; the resultant NO formation was abolished by the NOS inhibitor, L-NNA.

Ca2+ release and muscle cell contraction induced by cGMP and NO in permeabilized muscle cells were not affected by heparin, a blocker of IP3 receptors/Ca2+ channels (18, 24), or ruthenium red, a blocker of ryanodine receptors/Ca2+ channels (18, 19), whereas Ca2+ release induced by IP3 under similar conditions was abolished by heparin. The lack of effect of heparin or ruthenium red implied that cGMP did not cause Ca2+ release by activating IP3 receptors/Ca2+ channels or ryanodine receptors/Ca2+ channels. The latter are confined to smooth muscle cells isolated from the longitudinal layer, which, unlike smooth muscle cells from the circular layer, possess high-affinity binding sites for ryanodine and cyclic ADP-ribose but not for IP3 (18, 19). Furthermore, the effects of maximally effective concentrations of cGMP (10 µM) and IP3 (1 µM; 25) on Ca2+ release were approximately equal and additive, independently of which agent was applied first or of the interval separating application (2-10 min; Fig. 5). Together, IP3 and cGMP released ~60% of the Ca2+ stores. Addition of 10 µM ionomycin released a further 30% for a total of ~90% of Ca2+ store content. The pattern suggests that thapsigargin-sensitive Ca2+ stores discharged by IP3 and cGMP may be distinct, although possibly confluent, but it is not consistent with the notion that cGMP acted to enhance the activity of IP3-sensitive Ca2+ channels, since the ability of IP3 to induce Ca2+ release was not affected by the presence of cGMP (Fig. 5).

The mechanism mediating Ca2+ release by cGMP could be a cGMP-activated Ca2+ channel or a cGMP-inhibited sarcoplasmic Ca2+ pump. cGMP-gated cationic channels have been described in several locations [in retinal rods (6, 16) and olfactory neurons (30) and in pancreatic acinar cells (1, 31, 43)]. The retinal cGMP-gated cationic channel contains a cGMP-binding domain that possesses substantial homology to the cGMP-binding domain of PKG (16). The structure of the cGMP-gated Ca2+ channel in pancreatic acinar cells, thought to mediate sustained Ca2+ influx following agonist stimulation, has not been determined.

Sarcoplasmic Ca2+ channels and Ca2+ pumps are known to be susceptible to regulatory phosphorylation by PKA and PKG, providing a basis for the requirement of PKA and PKG inhibitors to unmask the Ca2+-mobilizing action of cGMP. Regulation of the IP3 receptor type I, which is predominantly expressed in vascular smooth muscle and cerebellum, is mediated by PKG-dependent phosphorylation at serine-1755 (11, 17). Our previous studies on gastric smooth muscle cells have shown that both PKA and PKG regulate IP3-dependent Ca2+ release and that PKG, in addition, stimulates Ca2+ uptake by the sarcoplasmic Ca2+ pump (27, 28). It seems possible, by analogy, that a cGMP-dependent Ca2+ release mechanism is regulated by PKG- and PKA-dependent phosphorylation.

Unlike smooth muscle where PKG acts to inhibit IP3- and cGMP-dependent Ca2+ release, Ca2+ release in other cell types appears to be indirectly mediated by PKG. Phosphorylation of the IP3 receptor in hepatocytes increases its sensitivity for Ca2+ release and induces oscillatory Ca2+ signals (36). Phosphorylation of ADP ribosyl cyclase by PKG results in synthesis of the Ca2+-mobilizing messenger, cyclic ADP-ribose; this mechanism is thought to underlie the ability of cGMP acting via PKG to initiate Ca2+ mobilization at fertilization in the sea urchin egg (12, 13). A similar mechanism has been claimed for NO-induced Ca2+ increase in interstitial cells of Cajal, but no direct evidence was provided for involvement of either cGMP or PKG (33).

The mechanism underlying the ability of cGMP to stimulate Ca2+ release in gastric smooth muscle is different from the mechanism underlying the ability of GTP to stimulate Ca2+ release in neuronal (NIE-115), smooth muscle (DDT1MF-2 and BC3H1), and fibroblast (WI-38) cell lines (5, 10, 14, 40). Ca2+ release induced by GTP in these cells is additive to that of IP3 and appears to be mediated by a product of GTP hydrolysis, since it could not be reproduced by nonhydrolyzable analogs of GTP (14). Ca2+ release could not be elicited also by cGMP in permeabilized muscle cells or microsomes, making it unlikely that a Ca2+-mobilizing action of cGMP in these cell lines was masked by activation of PKG. In the present study on freshly dispersed gastric smooth muscle cells, however, Ca2+ release was induced by cGMP in the absence of protein kinase activity but not by GTP.

The functional significance of a Ca2+-mobilizing action of cGMP may reside in greater Ca2+ requirements during development. Lincoln and Cornwell (21) have shown that repeated passage of vascular smooth muscle cells results in a substantial reduction in the expression of PKG. It is possible, although speculative, that PKG is either absent or minimally expressed during a stage in development when requirements for intracellular Ca2+ are high and could thus be met by a Ca2+-mobilizing action of cGMP that is additive to that of the usual messenger, IP3.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-28300 and DK-15564.

    FOOTNOTES

Address for reprint requests: G. M. Makhlouf, PO Box 980711, Medical College of Virginia, Richmond, VA 23298-0711.

Received 18 November 1997; accepted in final form 13 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bahnson, T. D., S. J. Pandol, and V. E. Dionne. Cyclic GMP modulates depletion-activated Ca2+ entry in pancreatic acinar cells. J. Biol. Chem. 268: 10808-10812, 1993[Abstract/Free Full Text].

2.   Beavo, J. A., M. Conti, and R. A. Heaslip. Multiple cyclic nucleotide phosphodiesterases. Mol. Pharmacol. 46: 399-405, 1994[Abstract].

3.   Bredt, D. S., and S. H. Snyder. Nitric oxide, a novel neuronal messenger. Neuron 8: 3-11, 1992[Medline].

4.   Chen, X.-L., and C. M. Rembold. Cyclic-nucleotide-dependent regulation of Mn2+ influx, [Ca2+]i, and arterial smooth muscle relaxation. Am. J. Physiol. 263 (Cell Physiol. 32): C468-C473, 1992[Abstract/Free Full Text].

5.   Chueh, S.-H., J. M. Mullaney, T. K. Ghosh, A. L. Zachary, and D. L. Gill. GTP- and inositol 1,4,5-trisphosphate-activated intracellular calcium movements in neuronal and smooth muscle cell lines. J. Biol. Chem. 262: 13857-13864, 1987[Abstract/Free Full Text].

6.   Cook, N. J., W. Hanke, and U. B. Kaupp. Identification, purification and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors. Proc. Natl. Acad. Sci. USA 84: 585-589, 1987[Abstract].

7.   Cornwell, T. L., E. Arnold, N. J. Boerth, and T. M. Lincoln. Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. Am. J. Physiol. 267 (Cell Physiol. 36): C1405-C1413, 1994[Abstract/Free Full Text].

8.   Cornwell, T. L., and T. M. Lincoln. Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells. Regulation of Ca2+ by atriopeptin and 8-bromo-cGMP is mediated by cGMP-dependent protein kinase. J. Biol. Chem. 264: 1146-1155, 1989[Abstract/Free Full Text].

9.   Cornwell, T. L., R. B. Pryzwansky, T. A. Wyatt, and T. M. Lincoln. Regulation of sarcoplasmic reticulum protein phosphorylation by localized cGMP-dependent protein kinase in vascular smooth muscle cells. Mol. Pharmacol. 40: 923-931, 1991[Abstract].

10.   Engling, R., K. J. Fohr, T. P. Kemmer, and M. Gratzl. Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells. Cell Calcium 12: 1-9, 1991[Medline].

11.   Firris, C. D., A. M. Cameron, D. S. Bredt, R. L. Huganir, and S. H. Snyder. Inositol 1,4,5-trisphosphate receptor is phosphorylated by cAMP-dependent protein kinase at serine 1755 and 1589. Biochem. Biophys. Res. Commun. 175: 192-198, 1991[Medline].

12.   Galione, A. Cyclic ADP-ribose, the ADP-ribosyl cyclase pathway and calcium signaling. Mol. Cell. Endocrinol. 98: 125-131, 1994[Medline].

13.   Galione, A., A. White, N. Wilmott, M. Turner, B. V. L. Potter, and S. P. Watson. cGMP mobilizes intracellular Ca2+ in sea urchin eggs by stimulating cyclic ADP-ribose synthesis. Nature 365: 456-459, 1993[Medline].

14.   Gill, D. L., T. Ueda, S.-H. Chueh, and M. W. Noel. Ca2+ release from endoplasmic reticulum by guanine nucleotide regulatory mechanism. Nature 320: 461-464, 1986[Medline].

15.   Jin, J.-G., K. S. Murthy, J. R. Grider, and G. M. Makhlouf. Stoichiometry of neurally induced VIP release, NO formation, and relaxation in rabbit and rat gastric muscle. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G357-G369, 1996[Abstract/Free Full Text].

16.   Kaupp, U. B., T. Niidome, T. Tanabe, S. Terada, W. Bongik, W. Stuhmer, N. J. Cook, K. Kanagawa, H. Matsuo, T. Hirose, T. Miyata, and S. Numa. Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature 342: 762-766, 1989[Medline].

17.   Komalavilas, P., and T. M. Lincoln. Phosphorylation of the inositol 1,4,5-trisphosphate receptor. Cyclic GMP-dependent protein kinase mediates cAMP- and cGMP-dependent phosphorylation in the intact rat aorta. J. Biol. Chem. 271: 21933-21938, 1996[Abstract/Free Full Text].

18.   Kuemmerle, J. F., and G. M. Makhlouf. Agonist-stimulated cyclic ADP ribose. Endogenous modulator of Ca2+-induced Ca2+ release in intestinal longitudinal muscle cells. J. Biol. Chem. 270: 25488-25494, 1995[Abstract/Free Full Text].

19.   Kuemmerle, J. F., K. S. Murthy, and G. M. Makhlouf. Agonist-mediated influx activates ryanodine-sensitive, IP3-insensitive Ca2+ release channels in longitudinal muscle of intestine. Am. J. Physiol. 266 (Cell Physiol. 35): C1421-C1431, 1994[Abstract/Free Full Text].

20.   Lee, H. C. Cyclic ADP-ribose: a new member of a super family of signaling cyclic nucleotides. Cell. Signal. 6: 591-600, 1994[Medline].

21.   Lincoln, T. M., and T. L. Cornwell. Intracellular cyclic GMP receptor proteins. FASEB J. 7: 328-338, 1993[Abstract/Free Full Text].

22.   Lincoln, T. M., M. Thompson, and T. L. Cornwell. Purification and characterization of two forms of cGMP-dependent protein kinase from bovine aorta. J. Biol. Chem. 263: 17263-17267, 1988.

23.   Mery, P. F., S. M. Lohmann, U. Walter, and R. Fischmeister. Ca2+ current is regulated by cGMP-dependent protein kinase in mammalian cardiac myocytes. Proc. Natl. Acad. Sci. USA 88: 1197-1201, 1991[Abstract].

24.   Murthy, K. S., J. R. Grider, and G. M. Makhlouf. InsP3-dependent Ca2+ mobilization in circular but not longitudinal muscle cells of intestine. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G937-G944, 1991[Abstract/Free Full Text].

25.   Murthy, K. S., J.-G. Jin, J. R. Grider, and G. M. Makhlouf. Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G1391-G1399, 1997[Abstract/Free Full Text].

26.   Murthy, K. S., and G. M. Makhlouf. VIP/PACAP-mediated activation of membrane-bound NO synthase in smooth muscle is mediated by pertussis toxin-sensitive Gi1-2. J. Biol. Chem. 269: 15977-15980, 1994[Abstract/Free Full Text].

27.   Murthy, K. S., and G. M. Makhlouf. Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cell. Am. J. Physiol. 268 (Cell Physiol. 37): C171-C180, 1995[Abstract/Free Full Text].

28.   Murthy, K. S., C. Severi, J. R. Grider, and G. M. Makhlouf. Inhibition of IP3 and IP3-dependent Ca2+ mobilization by cyclic nucleotides in isolated gastric muscle cells. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G967-G974, 1993[Abstract/Free Full Text].

29.   Murthy, K. S., K.-M. Zhang, J.-G. Jin, J. R. Grider, and G. M. Makhlouf. VIP-mediated G protein-coupled Ca2+ influx activates a constitutive NOS in dispersed gastric muscle cells. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G660-G671, 1993[Abstract/Free Full Text].

30.   Nakamura, T., and G. H. Gold. A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature 325: 442-444, 1987[Medline].

31.   Pandol, S. J., and M. S. Schoeffield-Payne. Cyclic GMP mediates the agonist-stimulated increase in plasma membrane calcium entry in the pancreatic acinar cell. J. Biol. Chem. 265: 12846-12853, 1990[Abstract/Free Full Text].

32.   Poggioli, J., and J. W. Putney, Jr. Net calcium fluxes in rat acinar cells: evidence for a hormone-sensitive calcium pool in or near the plasma membrane. Pflügers Arch. 292: 239-243, 1982.

33.   Publicover, N. G., E. M. Hammond, and K. M. Sanders. Amplification of nitric oxide signaling by interstitial cells isolated from canine colon. Proc. Natl. Acad. Sci. USA 90: 2087-2091, 1993[Abstract].

34.   Raeymaekers, L., F. Hoffman, and R. Casteels. Cyclic GMP-dependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle. Biochem. J. 252: 269-273, 1988[Medline].

35.   Rashatwar, S. S., T. L. Cornwell, and T. M. Lincoln. Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 84: 5685-5689, 1987[Abstract].

36.   Rooney, T. A., S. K. Joseph, C. Queen, and A. P. Thomas. Cyclic GMP induces oscillatory calcium signals in rat hepatocytes. J. Biol. Chem. 271: 19817-19825, 1996[Abstract/Free Full Text].

37.   Satake, N., S. Fujimoto, and S. Shibata. The potentiation of nitroglycerin-induced relaxation by PKG inhibition in rat aortic rings. Gen. Pharmacol. 27: 701-705, 1996[Medline].

38.   Smith, J. A., S. H. Francis, K. A. Walsh, S. Kumar, and J. D. Corbin. Autophosphorylation of type Ibeta cGMP-dependent protein kinase increases basal catalytic activity and enhances allosteric activation by cGMP and cAMP. J. Biol. Chem. 271: 20756-20762, 1996[Abstract/Free Full Text].

39.   Twort, C. H. C., and C. Van Breemen. Cyclic guanosine monophosphate-enhanced sequestration of Ca2+ by sarcoplasmic reticulum in vascular smooth muscle. Circ. Res. 62: 961-964, 1988[Abstract].

40.   Ueda, T., S. H. Chueh, M. W. Noel, and D. L. Gill. Influence of inositol 1.4.5-trisphosphate and guanine nucleotides in intracellular Ca2+ release within the NIE-115 neuronal cell line. J. Biol. Chem. 261: 3184-3192, 1986[Abstract/Free Full Text].

41.   Wojcikiewicz, R. J. H. Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J. Biol. Chem. 270: 11678-11683, 1995[Abstract/Free Full Text].

42.   Wolfe, L., J. D. Corbin, and S. H. Francis. Characterization of novel isozyme of cGMP-dependent protein kinase from bovine aorta. J. Biol. Chem. 264: 7734-7741, 1989[Abstract/Free Full Text].

43.   Xu, X., R. A. Star, G. Tortorici, and S. Muallem. Depletion of intracellular Ca2+ stores activates nitric-oxide synthase to generate cGMP and regulate Ca2+ influx. J. Biol. Chem. 269: 12645-12653, 1994[Abstract/Free Full Text].

44.   Yoshida, Y., H.-T. Sun, J.-Q. Cai, and S. Imai. Cyclic GMP-dependent protein kinase stimulates the plasma membrane Ca2+ pump ATPase of vascular smooth muscle via phosphorylation of a 240 kDa protein. J. Biol. Chem. 266: 19819-19825, 1991[Abstract/Free Full Text].


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