The norepinephrine-stimulated inositol phosphate response in human atria

J Mol Cell Cardiol 27, 2415-2419 (1995)

Brief Communication

The Norepinephrine-stimulated Inositol Phosphate Response in Human Atria Karen E. Anderson, Kim A. Lambert and Elizabeth A. Woodcock Baker Medical Research Institute, Commercial Road, Prahran, Vic, 3 1 8 1 Australia (Received 21 February 1995, accepted in revisedform 23 May 1995) K. E. ANDERSON, K. A. LAMBERT AND E. A. WOODCOCK.The Norepinephrine-stimulated Inositol Phosphate Response

in Human Atria. Journal of Molecular and Cellular Cardiology (1995) 27, 2415-2419. Inositol phosphate release and metabolism were studied in right atrial appendages obtained from 18 patients undergoing coronary artery bypass surgery and/or mitral valve replacement. [3H]Inositol-labeled human atria contained inositol(1,4, 5)trisphosphate, inositol(1,4)bisphosphate and the 1- (or 3) and 4-isomers of inositol monophosphate. Addition of norepinephrine (100/~mol/1) activated the release of inositol phosphates, as indicated by increased [3H]inositol label in all of these inositol phosphates. However, the phosphorylation product of inositol (1,4,5)trisphosphate, inositol-(1,3,4,5)tetrakisphosphate, and its metabolic products were not detected, either in control or stimulated atria. Similar inositol phosphate profiles were observed in rat right atria. Furthermore, both human and rat atria contained high concentrations of inositol(1,4,5)trisphosphate, which were not observed to increase with norepinephrine stimulation. The inositol phosphate responses to norepinephrine in rat and human cardiac tissue appear to be similar, except for the generally lower activity observed in human tissue. Thus, the rat provides a suitable model for the study of cardiac phosphatidylinositol turnover. © 1995 AcademicPress Limited KEY WORDS:Inositol phosphates: Human; Rat; Ins(1,4,5)P3

Introduction

of Ins(1,4, 5)P3 not observed, even under conditions of maximal prolonged stimulation. To explain these and other findings, we have proposed a model whereby stimulation of cq-adrenergic receptors causes primarily the hydrolysis of PtdIns(4)P to Ins(1,4,)P2, which is the source of most of the accumulated inositol phosphates (Woodcock et al., 1995). This unusual pathway may exist as a cardioprotective mechanism against possible patological consequences of Ins(1,4,5)P3 release (Anderson et al., 1995). The pathway in rat heart is unusual and it is important to establish whether similar properties are observed in cardiac tissue from other species, especially human. Previous studies have demonstrated inositol phosphate responses to endothelin-1 in h u m a n atria (Vogelsang et al., 1994) and to ~x-adrenergic receptor stimulation in h u m a n ventricular tissue (Bristow et al., 1989; Kohl et al., 1989), but in these studies, properties of the pathway were not ex-

In addition to the functionally predominant fl-adrenergic receptors, heart contains ~tl-adrenergic receptors, stimulation of which is associated with positive inotropy, atrial natriuretic peptide release, and hypertrophy (Benfey, 1993). Myocardial ~qadrenergic receptors, like those in other tissues, are linked to phosphatidylinositol (Ptdlns) turnover. In most cell types, this pathway involves the activation of protein kinase C together with raised cytosolic calcium, orchestrated by the generation of sn-l,2diacylglycerol and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) from phosphatidylinositol-(4,5)bisphosphate (PtdIns(4,5)P2) hydrolysis (Berridge, 1993). We have previously reported unusual properties of the PtdIns turnover pathway in intact rat heart preparations (Woodcock et al., 1987, 1995; Anderson et al., 1995), with phosphorylation products

Please address all correspondenceto: Dr E. A. Woodcock,Baker MedicalResearch Institute, CommercialRoad, Prahran, Vie,Australia. 0022-2828/95/102415 +05 $12.00/0

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amined in detail. In the current study, we have made a detailed analysis of inositol phosphate release and metabolism in norepinephrine stimulated human atrial tissue and compared findings with data obtained using rat atria.

Methods [3H]lnositol labeling of human atrial appendage pieces Studies performed complied with procedures approved by the Alfred Hospital Ethics Review Committee. Right atrial appendage tips, which were routinely amputated and discarded during cannulation of the heart for cardiopulmonary bypass, were obtained from 18 patients, nine male and nine female, aged between 44 and 81 years (mean___S.E.M, 60 + 3) during either coronary artery bypass graft surgery, and/or mitral valve replacement. None of the patients had apparent heart failure or had been given c~-adrenergic receptor antagonists, while eight were prescribed fl-adrenergic receptor antagonists. There was no observable difference in the Ptdlns pathway between patients receiving fl-adrenergic antagonists and those taking other medications. Appendages were excised immediately prior to atrial cannulation, removed to HEPES-buffered Krebs' solution on ice, and transported to the laboratory. Atrial appendages were then divided into thin pieces, 70-100mg in weight, in ice-cold medium, and suspended in water-jacketed organ baths containing HEPES-buffered Krebs' solution equilibrated in 5% CO2-95% O2, pH 7.4 at 32°C. Time from obtaining appendage to suspension in organ baths was less than 15 min. Following several washes, inositol phospholipids were labeled with [3H]inositol (40 pCi/rnl) for 5 h. Medium containing [3H]inositol was then removed, and replaced with medium containing propranolol (1 pmol/1) and LiCI (50 mmol/1) to block fl-adrenergic receptors and to inhibit inositol phosphate metabolism, respectively (Shears, 1992). After 10 rain incubation, atrial pieces were stimulated with norepinephrine (100 #mol/1) for the indicated times. Inositol phosphate release was terminated by freezing tissue in liquid N2.

into ice-cold HEPES-buffered Krebs' medium. Right atria were dissected and suspended in water jacketed organ baths as described for human atrial appendage.

Separation and quantitation of 3H-labeled inositol phosphates Inositol phosphates were extracted from frozen atrial samples in 2.0 ml ice-cold 5% trichloroacetic acid (TCA) containing 2.5mmol/1 EDTA and 5retool/1 phytic acid, as described previously (Woodcock et al., 1995). Inositol phosphates were separated by anion exchange high performance liquid chromatography (HPLC) using a Whatman Partisil (10 pro) SAX column in a Waters' Radial Compression Unit. A complex ammonium phosphate gradient (pH 3.8) from 0 to 2 mol/l was used, as described previously (Woodcock et al., 1995). 3HLabeled compounds were detected and quantitated using an on-line fl-counter (model CR, Radiomatic Instruments). All peaks were identified by comparison with commercial standards. The minimum peak detection level was 100 cpm.

Ins(1,4,5)P3 mass analysis Atrial extracts were prepared as described above except for the omission of [3H]inositol label during the 5-h incubation, and for the use of 5 mmol/1 ATP instead of phytic acid in the extraction procedure (Woodcock et al., 1995). Mass of Ins(1,4,5)P3 was measured using a competitive binding assay (Amersham TRK1000) essentially as described elsewhere (Divecha et al., 1991).

Statistics Values are presented as mean+s.E.M, n>_4. Statistical analyses was performed using Student's unpaired t-test for single comparisons, or one way analysis of variance (ANOVA) for multiple comparisons. Using either method, significance was defined as P
Materials 3H-labeling of rat right atria Male Sprague-Dawley rats of 250-3 O0 g were killed by decapitation and hearts removed immediately

[3H]Inositol, and assay kits for mass measurement of Ins(1,4,5)P3 were obtained from the Radiochemical Centre, Amersham, Bucks., IlK.

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Figure 1 Anion-exchange HPLC profiles of 3H-labeled inositol phosphates in human right atrial appendage (upper panel), and the effects of stimulation with norepinephrine (lower panel). Appendages were labeled with [3H]inositol for 5 h and subsequently stimulated for 20 min with norepinephrine (1 O0 I~mol/l)in the presence of propranolol (1/Jmol/l) and Li(50 mmol/1) as described in the "Methods" section. Control preparations were treated similarly except that norepinephrine was omitted.

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Results and Discussion Human right atrial appendage contained 3H-labeled inositol phosphates identified as Ins(1/3)P,, ins(4)P1, Ins(1,4)P2 and Ins(1,4,5)P3 (Fig. 1). Stimulation with maximally effective concentrations of norepinephrine (lO0/~mol/1) produced time-dependent increases in all of the detected inositol phosphates (Fig. 2a), indicating activation of the PtdIns turnover pathway. Twenty minutes norepinephrine stimulation resulted in a moderate enhancement of [3H]Ins(1,4,5)P3 accumulation, while larger increases in [3H]Ins(1,4)P2 and [3H]InsP1 were observed. Unlike most cell types, 3Hlabeled Ins(1,3,4,5)P4 and its dephosphorylation products, Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, could not be detected either in control or norepinephrine-stimulated atrial samples (Fig. 1). The

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pattern of inositol phosphate release was similar for all subjects, however the uptake and incorporation of [3H]inositol differed between individuals. When data were expressed as a percentage of control values, there was little variation between individual atria. 3H-labeled rat right atria also contained undetectable levels of Ins(1,3,4,5)P4 and its metabolic products, with only [3H]Ins(1,4.5)P3 and its dephosphorylation products Ins(1,4)P2 and the isomers of InsP, observed. Similar to findings in human atria, these inositol phosphates increased in the presence of norepinephrine (Fig. 2b), although only relatively small increases in Ins(1,4,5)P3 were observed. Stimulation of tissue at earlier time points of 15 and 30 s did not produce significant increases in any of the inositol phosphates detected (data not shown). We have previously reported similar unusual properties of inositol phosphate release and metabolism in intact rat left atria (Woodcock et al., 1995) and ventricular tissue (Anderson et al., 1995). Work of other laboratories has provided evidence that the pathway in guinea-pig atrium is similar to that in rat (Sakuma et al., 1988), and data from our laboratory indicate similar findings in mouse heart (unpublished data, 1994). Thus, an abbreviated phosphatidylinositol turnover pathway appears to be characteristic of heart tissue from a number of different species. In left atria this pathway is associated with high mass content of Ins(l,4, 5)P3 (13.5 _ 1.1 pmol/mg protein; Woodcock et al., 1995). Similar high concentrations of Ins(1,4,5)P3 have been demonstrated in adult rat ventricle (Anderson et al., 1995; Iwami et al., 1992) and neonatal ventricles (Fitzgerald et al., 1994), suggesting high mass content to be a property of rat heart. Accordingly, an investigation of the mass content of Ins(1,4,5)P3 in both rat and human right atria was performed. Human right atrial appendage contained high concentrations of Ins(1,4,5)P3 of l l . 4 + l . 2 p m o l / m g tissue which was not significantly altered by 20 rain norepinephrine stimulation (12.4 +_2. 7 pmol/mg tissue). Similarly, the content of Ins(1,4,5)P3 in rat right atria was high (12.6_ 0.5 pmol/mg protein) and unchanged with 20 rain norepinephrine (13.1 -I-2.8 pmol/mg protein). Thus, the observed increase in 3H-labeled Ins(1,4,5)P3 is a result of increased specific activity, reflecting the fact that the tissue was not labeled to equilibrium, as discussed in detail previously (Woodcock et al., 1995). Non-equilibrium labeling does not significantly alter inositol phosphate profiles as demonstrated previously in isolated neonatal myocytes (Woodcock et al., 1995). The profiles of inositol phosphate release in

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K.E. Anderson et al. Ca)

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Figure 2 Time course of accumulation of ~H-labeled inositol phosphates in human right atrial appendage (a) and rat right atria (b). Atria were treated as described in Figure 1. Atria were stimulated with norepinephrine (100 pmol/l) (O or Ira), or incubated in the absence of norepinephrine for 20 min (O or U]). Values shown in human atria (a) are a representative experiment, as different uptake and incorporation of 3H-inositol between individuals resulted in variable control levels. However the pattem of stimulation was similar between subjects, and when expressed as a percentage of control values, there was little variation [at 20 min norepinephrine stimulation 193 ___13 % for Ins( 1/3)P1, 233 _ 21% for Ins(4)P1, 286 +22% for Ins(1,4)P2, and 179 + 19% for Ins(1,4,5)P3, mean__+s.E.M., n>4]. Values shown in panel b are mean+s.E.M, of at least four separate determinations. Statistical analysis was performed by one-way ANOVA, *P<0.05 v unstimulated tissue. Squares represent Ins(4)Pl.

h u m a n and rat right atria appear to be similar. The only m a j o r difference between the two was the generally higher activity of the pathway in rat heart. This might be due to a lower concentration of cq-adrenergic receptors in h u m a n heart (Bristow et al., 1988), or possibly might reflect the fact that the h u m a n tissue was removed from patients undergoing bypass surgery and therefore could not be considered entirely healthy. On the basis of detailed mechanistic studies, we have proposed that the inositol phosphate response in rat left atria involves primarily the generation of Ins(1,4)P2, and that Ins(1,4,5)P3 release and metabolism is a minor component of the overall response (Woodcock et al., 1995). The quantitatively small response in h u m a n tissue precludes such detailed mechanistic studies. However, data in the present study suggest that the pathway in h u m a n and rat are essentially similar, with both species lacking detectable levels of Ins(1,3,4,5)P4, and containing high and unchanging concentrations of Ins(1,4,5)P3. Thus, the rat provides

a suitable model for study of h u m a n phosphatidylinositol turnover, and m a y provide insight into the role of the Ptdlns turnover pathway under both physiological and pathological conditions.

Acknowledgements This study was supported by the Australian National Health and Medical Research Council and by a grant-in-aid from the National Heart Foundation of Australia. Karen Anderson is a recipient of a Dora Lush Biomedical Postgraduate Research Scholarship. We thank Dr Franklin Rosenfeldt and Mr Bruce Davis of the Alfred Hospital for their assistance in supplying h u m a n tissue.

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Inositol Phosphates in Human Atria ischemia and reperfusion in the rat heart. Circ Res 76: 261-268. BENFEY BG, 1993. Function of myocardial a-adrenoceptors. ] Auton Pharmacol 1 3 : 3 5 1 - 3 7 2 . BERRIDCE MI, 1993. Inositol trisphosphate and calcium signalling. Nature 361: 315-325. BRISTOWMR, MINOBEW, RASMUSSENR, HERSHBERGERRE, HOFrMAN BB, 1988. Alpha-l-adrenergic receptors in the non failing and failing human heart, ] Pharmacol Exp Ther 247: 1039-1045. BRISTOW MR, 8ANDOVAL AB, GILBERT EM, DEISHER T, MONOBEVV, RASMUSSENR, 1989. Myocardial c~- and fladrenergic receptors in heart failure: Is cardiac-derived norepinephrine the regulatory signal? Eur Heart ] 9(H): 35--40. DIVECHA N, BANEIC H, IRVINE RF, 1991. The polyphosphoinositide cycle exists in the nuclei of swiss 3T3 cells under the control of a receptor (for IGF-1) in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and apparently induces translocation of protein kinase C to the nucleus. EMBO ] 10: 3207-3214. KOHL C, SCHMIT W, SCHOLZ H, 8CHOLZ J, TOTH M, DtJRING V, KALMARP, 1989. Evidence for cq-adrenoceptor-mediated increase of inositol trisphosphate in the human heart. ] Carddovasc Phannacol 13: 324-327. [WAMIG, TsuII Y, KAJIYAMAK, TSUTOMUO, SUFUH, 1992. Activation of PKC and IP3 during pressure overload

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