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J Biol Chem, Vol. 275, Issue 15, 10845-10850, April 14, 2000

Ca²⁺-activated but Not G Protein-mediated Inositol Phosphate Responses in Rat Neonatal Cardiomyocytes Involve Inositol 1,4,5-Trisphosphate Generation^*

Scot J. Matkovich and Elizabeth A. Woodcock

From the Cellular Biochemistry Laboratory, Baker Medical Research Institute, Melbourne 8008, Victoria, Australia

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inositol phosphate (InsP) responses to receptor activation are assumed to involve phospholipase C cleavage of phosphatidylinositol 4,5-bisphosphate to generate Ins(1,4,5)P₃. However, in [³H]inositol-labeled rat neonatal cardiomyocytes (NCM) both initial and sustained [³H]InsP responses to ₁-adrenergic receptor stimulation with norepinephrine (100 µM) were insensitive to the phosphatidylinositol 4,5-bisphosphate-binding agent neomycin (5 mM). Introduction of 300 µM unlabeled Ins(1,4,5)P₃ into guanosine 5'-3-O-(thio)triphosphate (GTPS)-stimulated, permeabilized [³H]inositol-labeled NCM increased [³H]Ins(1,4,5)P₃ slightly but did not significantly reduce levels of its metabolites [³H]Ins(1,4)P₂ and [³H]Ins(4)P, suggesting that these [³H]InsPs are not formed principally from [³H]Ins(1,4,5)P₃. In contrast, the calcium ionophore A23187 (10 µM) provoked [³H]InsP responses in intact NCM which were sensitive to neomycin, and elevation of free calcium in permeabilized NCM led to [³H]InsP responses characterized by marked increases in [³H]Ins(1,4,5)P₃ (2.9 ± 0.2% of total [³H]InsPs after 20 min of high Ca²⁺ treatment in comparison to 0.21 ± 0.05% of total [³H]InsPs accumulated after 20 min of GTPS stimulation). These data provide evidence that Ins(1,4,5)P₃ generation is not a major contributor to G protein-coupled InsP responses in NCM, but that substantial Ins(1,4,5)P₃ generation occurs under conditions of Ca²⁺ overload. Thus in NCM, Ca²⁺-induced Ins(1,4,5)P₃ generation has the potential to worsen Ca²⁺ overload and thereby aggravate Ca²⁺-induced electrophysiological perturbations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃),¹ a Ca²⁺-mobilizing second messenger, is critical in mediating responses to hormone stimulation in a wide variety of cell types (1). Activation of phospholipase C (PLC) generates both Ins(1,4,5)P₃ and sn-1,2-diacylglycerol from the precursor phospholipid PtdIns(4,5)P₂. However, all known PLC isozymes have the capacity to hydrolyze PtdIns and PtdIns(4)P as well as PtdIns(4,5)P₂, although with lower potency (2, 3). While there is some difference in substrate selectivity between the three PLC classes (, , and ), activity toward PtdIns(4,5)P₂ is somewhat higher than toward PtdIns(4)P when measurements are made using PLCs reconstituted in lipid vesicles (2, 3). Nonetheless, this suggests the possibility that cleavage of PtdIns(4)P in addition to PtdIns(4,5)P₂ could be a substantial contributor to the overall InsP response. Early studies of InsP responses led to the suggestion that generation of Ins(1,4,5)P₃ takes place during the initial stage of agonist stimulation, while the sustained InsP response depends on cleavage of PtdIns to Ins(1)P (4-6). However, the lack of potent, readily available inhibitors of Ins(1,4,5)P₃ metabolism has meant that such ideas have never been substantiated, and Ins(1,4,5)P₃ has generally been assumed to be the primary source of all InsPs that accumulate in response to receptor stimulation, including InsPs generated in the myocardium.

Beat-to-beat regulation of cardiac muscle Ca²⁺ is primarily controlled by ryanodine receptors on the sarcoplasmic reticulum. Ins(1,4,5)P₃-mediated Ca²⁺ responses in cardiac myocytes are relatively slow and weak and have been characterized as Ca²⁺ oscillations (7, 8) which have potential proarrhythmic activity. Intracellular application of Ins(1,4,5)P₃ causes action potential prolongation and degeneration (9) including activation of proarrhythmic sodium-calcium exchange (10). Furthermore, arrhythmias occurring during ischemia and reperfusion correlate with cardiac Ins(1,4,5)P₃ content (11-17). In the face of evidence for a pathological role of Ins(1,4,5)P₃ in the myocardium (reviewed in Ref. 18), it is important to better understand the mechanisms responsible for its generation in cardiac myocytes and also the mechanisms suppressing its generation under physiological circumstances.

Rat neonatal cardiomyocytes (NCM) represent a convenient system for the modelling of myocardial signaling. InsP responses to norepinephrine (NE) in myocytes are mediated via ₁-adrenergic receptors, coupling via G_q to PLC- isoforms (19, 20) of which PLC-1 and PLC-3 are expressed in heart (21-23). Our initial studies indicated that NE stimulation in NCM was largely insensitive to the PtdIns(4,5)P₂-binding agent neomycin (24), unlike findings in other cell types, implying that responses are largely independent of Ins(1,4,5)P₃. This prompted us to examine the source of InsPs in NCM generated in response to ₁-adrenergic activation. In this study, we present the first direct evidence that G protein activation of NCM stimulates an InsP response that is largely independent of Ins(1,4,5)P₃ generation. In contrast, the InsP response to elevation of intracellular Ca²⁺ is primarily via Ins(1,4,5)P₃.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Culture of Neonatal Cardiomyocytes-- NCM were prepared from 1- to 3-day-old Sprague-Dawley rat pups, essentially as described previously (25). NCM were isolated by repeated trypsin digestion with gentle mechanical dispersion, pre-plated twice for 30 min each to remove nonmyocytes and left to attach for 18 h in medium 199, 10% fetal calf serum, 0.1 mM bromodeoxyuridine, 50 units/ml penicillin G, and 50 µg/ml streptomycin sulfate onto uncoated dishes, at a typical seeding density of 700 cells/mm². Medium was then replaced with a defined serum-free medium consisting of medium 199, 2% (w/v) bovine serum albumin, 10 µg/ml human insulin, 10 µg/ml bovine apo-transferrin, 0.1 mM bromodeoxyuridine, 50 units/ml penicillin G, and 50 µg/ml streptomycin sulfate. Bromodeoxyuridine was omitted after 3 days and cells were labeled with [³H]inositol in defined serum-free medium for 48 h prior to experiments. Wells were washed extensively with nonradioactive medium before use.

Saponin Permeabilization of NCM-- Cell permeabilization was achieved by treatment with 50 µg/ml saponin in an intracellular-like medium containing an ATP-generating system for 4 min at 37 °C. Intracellular medium had the following constituents (in mM): KCl, 110; NaCl, 10; KH₂PO₄, 1; MgSO₄, 5; EGTA, 1; Hepes, 20; ATP, 2; creatine phosphate, 10; with 20 Sigma units/ml creatine phosphokinase, 0.2% (w/v) bovine serum albumin and CaCl₂ to give a free [Ca²⁺] of 100 nM (determined by reference to a Ca²⁺-EGTA titration curve), adjusted to pH 7.2 with NaOH. Plates were washed 4 times with intracellular medium and fresh intracellular medium was added before the commencement of [³H]InsP generation protocols.

Generation and Extraction of [³H]InsPs-- 10 mM LiCl was added 10 min before the addition of an appropriate stimulator to intact NCM in medium 199, or permeabilized NCM in intracellular medium with an ATP-generating system, and maintained during the treatment period in order to inhibit InsP breakdown by inositol polyphosphate-1-phosphatase and inositol monophosphatase (26, 27). 1 µM Propranolol was also added to intact NCM to block -adrenergic receptors. For stimulation of intact NCM, stock solutions of 10 mM NE in 0.5 M 2-mercaptoethanol (to inhibit catecholamine oxidation) or 10 mM A23187 in dimethyl sulfoxide were prepared freshly on the experimental day and diluted into medium 199 containing LiCl and propranolol just before use. After stimulation, medium was removed from intact NCM and the reaction terminated by the addition of ice-cold 5% trichloroacetic acid, 2.5 mM disodium EDTA, 5 mM sodium phytate (sodium inositol hexakisphosphate). Permeabilized NCM reactions were stopped by the addition of an equal volume of ice-cold 10% trichloroacetic acid, 5 mM disodium-EDTA, 5 mM sodium phytate to the intracellular medium. Wells were scraped, washed once with 5% trichloroacetic acid, 2.5 mM disodium EDTA, 5 mM sodium phytate and the extracts collected. Trichloroacetic acid-insoluble material was removed by low-speed centrifugation, a 1:1 mixture of freon/tri-n-octylamine was added to remove trichloroacetic acid, and the upper aqueous phase containing [³H]InsPs was prepared for high performance liquid chromatography as described previously (28).

Analysis of [³H]InsP Isomers-- Separation and quantitation of [³H]InsP isomers was performed using established anion-exchange high performance liquid chromatography techniques (28, 29). Briefly, ³H-labeled compounds were eluted with a complex gradient of ammonium phosphate at pH 3.8. Retention times and integrated peak values were obtained via an on-line -counter. Identification of [³H]InsP isomers was made with reference to appropriate commercial standards.

Materials-- Fetal calf serum was specially selected for low endotoxin and obtained from the Commonwealth Serum Laboratories, Parkville, Australia. Medium 199, Hepes, L-glutamine, bovine serum albumin, NaHCO₃, and other materials for the preparation of cell culture solutions and media were cell culture grade, obtained from Sigma and dissolved in highly purified Milli-Q H₂O. Human insulin was Actrapid^TM from Novo Nordisk Pharmaceuticals, and bovine apo-transferrin was from Sigma. GTPS and Ins(1,4,5)P₃ were obtained through Sigma and Sapphire Bioscience, Australia. [³H]Ins(1,4,5)P₃ (21.00 Ci/mmol) was purchased from NEN Life Science Products Inc. and [³H]inositol (18.00 Ci/mmol) was obtained from Amersham Pharmacia Biotech. Other reagents were obtained from Sigma or BDH/AnalaR and were of analytical reagent grade.

Treatment of Data-- Differences between treatment groups were assessed by unpaired Student's t test or 1-way ANOVA as appropriate, and accepted as statistically significant at p < 0.05. Unless otherwise noted, results shown are from representative experiments, which were independently repeated three times.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Time Course of NE and A23187 Stimulation of InsP Generation-- NCM were used for [³H]InsP generation experiments at 5 days after initial isolation and radiolabeled with 20 µCi/ml [³H]inositol 48 h before experiments. As it has been suggested that short- and long-term stimulation of PLC have different characteristics, we examined changes in individual [³H]InsP isomers in detail over the first minute of PLC stimulation and compared this to a 20-min stimulation period. After a 10-min pretreatment period with 10 mM LiCl and 1 µM propranolol in medium 199, PLC was activated by stimulation of ₁-adrenergic receptors with 100 µM NE, or by raising intracellular Ca²⁺ by treatment with 10 µM A23187, in the continued presence of LiCl and propranolol, for the specified time periods.

In agreement with previous reports (30, 31) we found a transient increase in [³H]Ins(1,4,5)P₃ followed by a decrease during the first minute of NE stimulation (Fig. 1). Stimulation by A23187 followed a similar pattern, although the response was of lower magnitude. From 1 to 20 min, further increases in [³H]Ins(1,4,5)P₃ were only marginal. As attack by 5-phosphatase and 3-kinase enzymes on Ins(1,4,5)P₃ is not inhibited by Li⁺, this observation alone does not reflect the amount of [³H]Ins(1,4,5)P₃ which could have been generated by PLC activity over this time frame.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Time course of NE and A23187 responses. [3H]Inositol-labeled NCM were incubated in medium 199 containing 10 mM LiCl and 1 µM propranolol for 10 min. 100 µM NE or 10 µM A23187 was then added for the indicated time period in the continued presence of LiCl and propranolol. In the absence of agonist, [3H]InsPs did not rise detectably during the first minute and at 20 min, unstimulated total [3H]InsPs increased to 4.3 × 104 cpm/well. Circles represent NE stimulation and triangles represent A23187 stimulation. Results shown are mean values from a representative experiment of three performed in duplicate.

Levels of [³H]Ins(1,4)P₂ and its immediate dephosphorylation product [³H]Ins(4)P followed a similar rise and fall to [³H]Ins(1,4,5)P₃ during the first minute of stimulation (Fig. 1), but continued to increase steadily thereafter in the presence of Li⁺, an uncompetitive inhibitor of Ins(1,4)P₂ and Ins(4)P breakdown (26, 32). As [³H]Ins(1,4)P₂ could be produced either by PLC hydrolysis of [³H]PtdIns(4)P or by breakdown of [³H]Ins(1,4,5)P₃ via a 5-phosphatase, these data demonstrate continued PLC activity over a 20-min period but do not indicate whether the primary product of PLC hydrolysis of phosphoinositides is Ins(1,4)P₂ or Ins(1,4,5)P₃.

Neomycin Sensitivity of NE and A23187 Responses-- 30 s of treatment with either NE or A23187 stimulated overall [³H]InsP generation (Fig. 2, a and b) and increased [³H]Ins(1,4,5)P₃ (Fig. 1). However, the inclusion of 5 mM neomycin, an aminoglycoside that binds to PtdIns(4,5)P₂ (24) and inhibits production of Ins(1,4,5)P₃ (33, 34) did not reduce [³H]InsP accumulation in response to NE either at 30 s (Fig. 2a) or at 20 min (Fig. 2c).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of neomycin on stimulated [3H]InsP responses. [3H]Inositol-labeled NCM were washed with medium 199 and incubated with fresh medium containing 10 mM LiCl and 1 µM propranolol for 10 min with or without 5 mM neomycin. 100 µM NE or 10 µM A23187 was then added for 30 s or 20 min. Open bars represent timed control, filled bars represent NE (panels a and c) or A23187 (panels b and d), and hatched bars represent NE or A23187 with neomycin. Results are presented as mean ± S.E. of a representative experiment of three performed in triplicate. * signifies p < 0.05 between treatments with or without neomycin.

In contrast, when NCM were stimulated with A23187 for either 30 s (Fig. 2b) or 20 min (Fig. 2d), 5 mM neomycin caused ~50% inhibition of the response. Approximately 50% inhibition by this concentration of neomycin has been reported in a number of different cell types (33, 34). Addition of neomycin in the absence of agonist had no effect on unstimulated [³H]InsPs (data not shown).

Ca²⁺-stimulated and G Protein-activated [³H]InsP Responses in Permeabilized NCM-- [³H]Inositol-labeled NCM were permeabilized, pretreated for 10 min with 10 mM LiCl, and [³H]InsP responses were stimulated by adding 10 µM GTPS, or AlF₄ (to activate heterotrimeric G proteins), or by increasing free [Ca²⁺] from 100 nM to 10 µM, in the continued presence of Li⁺. [³H]InsP generation was measured after 1 or 20 min of stimulation to examine both the early and sustained phase of [³H]InsP generation, as in intact NCM experiments.

10 µM GTPS did not cause a detectable increase in total [³H]InsPs or in [³H]Ins(1,4,5)P₃ at 1 min (data not shown). To ensure this did not reflect slow guanine nucleotide exchange on G_q (35), the experiments were repeated using AlF₄ which causes an immediate activation of G_q, independent of GDP/GTP exchange (20, 36). After 1 min of AlF₄ treatment, total [³H]InsP content was not significantly increased compared with control (Fig. 3b), and there was no increase in [³H]Ins(1,4,5)P₃ (Fig. 3a). 20 min of G protein stimulation with GTPS led to an approximate doubling of total [³H]InsPs (Fig. 3d), again without detectable increase in [³H]Ins(1,4,5)P₃ (Fig. 3c).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   [3H]InsP generation in response to elevated free Ca2+ and G protein stimulation in permeabilized NCM. [3H]Inositol-labeled, permeabilized NCM were pretreated for 10 min with Li+ at a free [Ca2+] of 100 nM and then exposed to Li+ for a further 1 or 20 min (open bars), treated with AlF4 (20 µM AlCl3, 5 mM sodium fluoride) for 1 min (panels a and b) or with 10 µM GTPS for 20 min (panels c and d) (hatched bars), or treated with 10 µM free Ca2+ for 1 or 20 min (filled bars). Results are presented as mean ± S.E. of a representative experiment of three performed in triplicate. * signifies p < 0.05 versus no additions.

In contrast to 1 min of G protein activation, when elevated [Ca²⁺] was used to activate [³H]InsP generation for 1 min, total [³H]InsPs were significantly increased (Fig. 3b). This stimulation was characterized by a very marked rise in the level of [³H]Ins(1,4,5)P₃ (Fig. 3a). Although total [³H]InsPs in response to 20 min of 10 µM Ca²⁺ were only slightly increased above those seen with 20 min of 100 nM Ca²⁺ (Fig. 3d), the rise in [³H]Ins(1,4,5)P₃ was sustained, with the level of [³H]Ins(1,4,5)P₃ at 20 min of 10 µM Ca²⁺ remaining significantly higher than that at 20 min of 100 nM Ca²⁺ (Fig. 3c). Thus, unlike the sustained effect of GTPS, the [³H]InsP response to elevated Ca²⁺ involved significant increases in [³H]Ins(1,4,5)P₃.

Blockade of [³H]Ins(1,4,5)P₃ Breakdown during G Protein-activated [³H]InsP Generation in Permeabilized NCM-- The low sensitivity to neomycin in NE-stimulated NCM (together with demonstrable neomycin inhibition of A23187 responses) suggested that the major proportion of InsPs detected upon G protein activation of phospholipase C do not arise from generation of Ins(1,4,5)P₃ and its subsequent breakdown. In order to investigate this further, and considering that permeabilized NCM experiments showed similar effects of G protein activation and Ca²⁺ elevation to those observed in intact NCM, we attempted to block metabolism of any [³H]Ins(1,4,5)P₃ generated in permeabilized NCM to ascertain its contribution to the overall [³H]InsP response. As the K_m of the enzymes responsible for Ins(1,4,5)P₃ metabolism is in the order of 10 µM (27, 37) 300 µM unlabeled Ins(1,4,5)P₃ was added to the medium to competitively inhibit breakdown of [³H]Ins(1,4,5)P₃. If other [³H]InsPs detected in permeabilized NCM arise from metabolism of [³H]Ins(1,4,5)P₃, this maneuver would be expected to increase the amount of [³H]Ins(1,4,5)P₃ detected at the expense of labeled metabolites.

Fig. 4 shows the effects of addition of 300 µM Ins(1,4,5)P₃ to unstimulated and GTPS-stimulated permeabilized NCM. When 10 µM GTPS was added to permeabilized NCM for 20 min, there was a significant increase in total [³H]InsPs consisting primarily of [³H]Ins(1,4)P₂ and [³H]Ins(4)P but not [³H]Ins(1,4,5)P₃. In the presence of 300 µM Ins(1,4,5)P₃, GTPS-stimulated total [³H]InsPs were unchanged, as expected. Only a small amount of [³H]Ins(1,4,5)P₃ was detected in the presence of 300 µM Ins(1,4,5)P₃ and GTPS, which was not observed with 300 µM Ins(1,4,5)P₃ alone. Experiments performed using AlF₄ in place of GTPS gave similar results (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Lack of effect of 300 µM Ins(1,4,5)P3 on G protein-stimulated [3H]InsP generation. [3H]Inositol-labeled, permeabilized NCM were treated for 10 min with 10 mM LiCl, then intracellular medium containing LiCl was renewed for a further 20-min period, in the absence of stimulus (open bars), or with the addition of 10 µM GTPS (filled bars), or with the addition of 300 µM Ins(1,4,5)P3 and 10 µM GTPS (striped bars), or with the addition of 300 µM Ins(1,4,5)P3 alone (hatched bars). A representative experiment of two performed in triplicate is shown, and an experiment was also performed using AlF4 in place of GTPS with identical results. * indicates significant difference between GTPS treatment and no additions, and  indicates significant difference between Ins(1,4,5)P3 addition and Ins(1,4,5)P3 + GTPS treatment (p < 0.05).

To confirm that [³H]Ins(1,4,5)P₃ could be successfully protected by the addition of excess unlabeled Ins(1,4,5)P₃ in NCM, we added 10 nM [³H]Ins(1,4,5)P₃ to permeabilized NCM not previously labeled with [³H]inositol, in the absence or presence of 300 µM Ins(1,4,5)P₃ together with 10 mM LiCl for 20 min. As shown in Fig. 5, 300 µM Ins(1,4,5)P₃ protected [³H]Ins(1,4,5)P₃ from dephosphorylation to [³H]Ins(1,4)P₂.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Protection of [3H]Ins(1,4,5)P3 by 300 µM unlabeled Ins(1,4,5)P3. Permeabilized, unlabeled NCM were incubated for 10 min with 10 mM LiCl, followed by addition of [3H]Ins(1,4,5)P3 (10 nM) in the absence or presence of 300 µM unlabeled Ins(1,4,5)P3 for 20 min. The major inositol phosphates recovered were [3H]Ins(1,4,5)P3 and [3H]Ins(1,4)P2. Open bars indicate addition of 10 nM [3H]Ins(1,4,5)P3 alone, solid bars indicate addition of 10 nM [3H]Ins(1,4,5)P3 and 300 µM unlabeled Ins(1,4,5)P3. * indicates significant difference between treatments (p < 0.01). Results are derived from two separate experiments performed in triplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study challenges the assumption that InsP responses in NCM depend on the primary generation of Ins(1,4,5)P₃, followed by its metabolism to generate a range of other InsPs. We provide evidence for generation of Ins(1,4,5)P₃ in response to elevation of intracellular Ca²⁺, as shown by the marked increase in Ins(1,4,5)P₃ observed during the response to elevated free Ca²⁺ in permeabilized NCM and by the sensitivity of A23187 stimulation to the PtdIns(4,5)P₂-binding aminoglycoside neomycin in intact NCM. In contrast to Ca²⁺-activated responses, ₁-adrenergic receptor stimulation leads to InsP generation which is relatively insensitive to neomycin, and direct G protein stimulation by either GTPS or AlF₄ initiates InsP responses which are largely independent of Ins(1,4,5)P₃.

Early studies in non-cardiac cell types suggested that there is a change in the preferred substrate for phospholipase C away from PtdIns(4,5)P₂ as agonist stimulation is maintained, such that Ins(1,4,5)P₃ generation predominates only in the initial phase of the response (5, 6). In NCM at both the 30-s and 20-min time points, the total [³H]InsP response to A23187 was inhibited by neomycin, indicating Ins(1,4,5)P₃ generation. In contrast, the response to NE was insensitive to neomycin at both time points. Thus, although time course data showed increased [³H]Ins(1,4,5)P₃ early in both the NE and A23187 responses, only the A23187 response was neomycin-sensitive in terms of total [³H]InsPs generated. This shows that a finding of increased [³H]Ins(1,4,5)P₃ cannot be taken alone as evidence that it is the primary source of accumulated [³H]InsPs, even at an early time point.

Similar insensitivity of NE responses to neomycin has been observed in adult rat cardiomyocytes (38) as well as normoxic preparations of adult rat heart tissue (29) but InsP responses under reperfusion conditions, during which large increases in Ins(1,4,5)P₃ are observed, are effectively blocked by aminoglycosides (13, 14, 17). Increases in levels of all InsP isomers are inhibited by aminoglycosides under reperfusion conditions, not merely Ins(1,4,5)P₃. Although neomycin at high concentrations may well have cellular effects unrelated to its PtdIns(4,5)P₂-binding capability, the differential sensitivity of the Ca²⁺ and NE responses to neomycin argues against a nonspecific action in this case. Thus, these data suggest that increased cytosolic Ca²⁺ is a stimulus for Ins(1,4,5)P₃ generation, whereas ₁-adrenergic receptor stimulation of PLC triggers an InsP response that is largely independent of PtdIns(4,5)P₂ hydrolysis.

As there are several G protein-coupled receptor systems present in NCM that could activate PLC, we chose to examine InsP responses to direct G protein activation in permeabilized NCM to test whether the relative incapacity of ₁-adrenergic receptor stimulation to induce sustained Ins(1,4,5)P₃ generation extended to all G protein-mediated PLC activities in NCM. Neither short-term (1 min) nor long-term (20 min) G protein stimulation was able to significantly increase [³H]Ins(1,4,5)P₃ content in permeabilized NCM even though total [³H]InsPs were increased at 20 min. However, this did not reflect a fundamental inability of the permeabilized NCM preparation to hydrolyze [³H]PtdIns(4,5)P₂, as elevation of free Ca²⁺ in the medium for either 1 or 20 min led to a dramatic increase in the amount of [³H]Ins(1,4,5)P₃ recovered. The steady increase of [³H]InsPs following G protein activation contrasted with the response to elevated free Ca²⁺, in which both [³H]Ins(1,4,5)P₃ and total [³H]InsPs increased rapidly up to 1 min, but the response was not sustained for 20 min.

The use of permeabilized NCM provided the opportunity to directly measure the contribution of [³H]Ins(1,4,5)P₃ generation to G protein-mediated [³H]InsP responses by competing for its breakdown with a large molar excess of unlabeled Ins(1,4,5)P₃. The inability of 300 µM Ins(1,4,5)P₃ to reduce the levels of unstimulated and GTPS-stimulated [³H]Ins(1,4)P₂ and [³H]Ins(4)P implies that [³H]Ins(1,4,5)P₃ is not the source of these [³H]InsPs. As the major labeled inositol phosphate which accumulates is [³H]Ins(4)P, which can only be produced by dephosphorylation of [³H]Ins(1,4)P₂, the most likely progenitor of InsPs in NCM is Ins(1,4)P₂ derived from PLC hydrolysis of PtdIns(4)P, as suggested in adult rat heart (29). Direct hydrolysis of PtdIns, which has been reported as an alternative to PtdIns(4,5)P₂ hydrolysis (4, 5, 33) would lead to increases in the levels of Ins(1)P rather than Ins(4)P. Increases in [³H]Ins(1)P or [³H]Ins(3)P (which cannot be resolved by high performance liquid chromatography) were only minor in comparison to increases in [³H]Ins(4)P observed during the [³H]InsP responses in either intact or permeabilized NCM described in the present study.

The InsP generation model suggested by these experiments allows for production of sn-1,2-diacylglycerol without concomitant generation of Ins(1,4,5)P₃ in response to G protein PLC-mediated stimuli. Activation of conventional and novel PKC isoforms in heart has been reported under a number of conditions, especially in relation to hypertrophic growth (39-41). Suppression of Ins(1,4,5)P₃ generation under physiological conditions in NCM, as previously suggested for adult rat heart (29), lends support to the view that increases in Ins(1,4,5)P₃ are deleterious to the heart (18). Direct intracellular application of Ins(1,4,5)P₃ causes action potential degeneration (9, 10) and Ins(1,4,5)P₃ generation has been shown to be related to the initiation of arrhythmias under ischemia and reperfusion conditions (11-17). It is therefore possible that the abbreviated InsP pathway present in cardiac myocytes protects the heart from Ins(1,4,5)P₃ generation and consequent arrhythmias, presumably arising from enhanced Ca²⁺ release from the cardiac sarcoplasmic reticulum. If this is the case, then it is interesting that Ca²⁺ overload itself causes Ins(1,4,5)P₃ generation, potentially initiating a positive feedback relationship between Ca²⁺ and Ins(1,4,5)P₃ which would further aggravate any electrophysiological damage to the heart.

An alternative explanation for preferential Ins(1,4)P₂ generation in the heart is that Ins(1,4)P₂ itself has a signaling function. While Ins(1,4)P₂ is generally considered to be an inactive metabolite of Ins(1,4,5)P₃, there have been a number of studies suggesting that it has a role in cellular growth (42, 43) and other metabolic pathways (44, 45). Clearly, further investigation would be required to establish any such role in the heart.

The mechanisms underlying the different responses to NE and Ca²⁺ remain to be determined. Whereas PLC- isoforms respond to G protein activation (46), PLC- isoforms (particularly PLC-1) are likely mediators of Ca²⁺-activated InsP responses in the absence of other upstream signaling molecules (46, 47). Surprisingly, PLC- has somewhat less specificity for PtdIns(4,5)P₂ relative to PtdIns(4)P than does PLC- when studied in vitro using synthetic lipid micelles (2). It is, however, possible that the substrate specificities of the different PLC isoforms might be altered by particular features of the cardiac sarcolemma.

In summary, the present study demonstrates that generation of Ins(1,4,5)P₃ in NCM requires Ca²⁺-mediated activation of PLC and that G protein activation of PLC causes an InsP response which is largely independent of Ins(1,4,5)P₃ generation. Further studies will be required to elucidate the molecular events responsible for the selection between Ins(1,4)P₂ and Ins(1,4,5)P₃ generation in NCM.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Baker Medical Research Institute, P. O. Box 6492, St. Kilda Rd. Central, Melbourne 8008, Victoria, Australia. Tel.: 61-3-9522-4333; Fax: 61-3-9521-1362; E-mail: liz.woodcock@baker.edu.au.

	ABBREVIATIONS

The abbreviations used are: Ins(1, 4,5)P₃, D-myo-inositol 1,4,5-trisphosphate; Ins(1, 4)P₂, D-myo-inositol 1,4-bisphosphate; Ins(4)P, D-myo-inositol 4-monophosphate; Ins(1)P, D-myo-inositol 1-monophosphate; InsP, inositol phosphate; PtdIns(4, 5)P₂, phosphatidylinositol 4,5-bisphosphate; PtdIns(4)P, phosphatidylinositol 4-monophosphate; PLC, phospholipase C; PKC, protein kinase C; NE, norepinephrine; GTPS, guanosine 5'-3-O-(thio)triphosphate; NCM, neonatal rat cardiomyocytes.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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