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(Circulation Research. 1996;78:806-812.)
© 1996 American Heart Association, Inc.

Articles

Identification of Protein Kinase C Isoforms in Rat Mesenteric Small Arteries and Their Possible Role in Agonist-Induced Contraction

Vasken Ohanian, Jacqueline Ohanian, Linda Shaw, Sylvia Scarth, Peter J. Parker, Anthony M. Heagerty

From the Department of Medicine (V.O., J.O., L.S., S.S., A.M.H.), Manchester (UK) Royal Infirmary, and the Protein Phosphorylation Laboratory (P.J.P.), Imperial Cancer Research Fund, London, UK.

Correspondence to Dr V. Ohanian, Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, England.

	Abstract

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Abstract We have identified immunologically the protein kinase C (PKC) isoforms present in rat mesenteric small arteries, defined their distribution between particulate and soluble fractions, and studied their involvement in phorbol ester–induced contraction. Our analysis revealed the presence of the Ca²⁺-dependent PKCs ( and ), Ca²⁺-independent PKCs ( and ), and the atypical isoform (). PKCß could not be detected, whereas PKC is likely to be of neural origin. All isoforms exhibited different distributions: PKC, PKC, and PKC were found in both particulate and soluble fractions. In contrast, PKC was mainly in the particulate fraction, and PKC was in the soluble fraction. Phorbol esters, which activate PKC and cause smooth muscle contraction, downregulated only the and isoforms. This was associated with a parallel loss of contractile response to phorbol ester. The force developed to submaximal concentrations of noradrenaline was decreased after phorbol dibutyrate pretreatment, although the sensitivity and maximal response were unchanged. Phorbol ester pretreatment did not affect the contractile response to vasopressin. The sensitivity to non–receptor-mediated contraction, caused by K⁺ in the presence of prazosin, was slightly reduced by 4- and 4ß-phorbol ester pretreatment. Maximal tension in response to this agonist was not affected. We conclude that PKC and/or PKC is necessary for phorbol ester–mediated contraction but is not essential for noradrenaline-, vasopressin-, or K⁺-induced contraction, demonstrating differences in the mechanisms involved in the contractile response between these agents.

Key Words: protein kinase C • phorbol ester • contraction • small arteries • vascular smooth muscle

	Introduction

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The regulation of blood flow by small arteries and their response to vasoconstrictor hormones are fundamental to the control of vascular resistance. Consequently, the cellular processes involved in vascular smooth muscle contraction have attracted considerable interest.

Vasoconstrictor hormone receptors belong to the family of seven transmembrane segment receptors, which are G protein–coupled to phosphoinositide–phospholipase C such that their activation by agonists such as angiotensin II and AVP causes a rapid hydrolysis of inositol phospholipids, resulting in the generation of two second messengers, IP₃ and DAG.¹ The former mobilizes intracellular Ca²⁺ to activate PKC as well as Ca²⁺/calmodulin-dependent kinases, with the subsequent phosphorylation of myosin light chain and the initiation of contraction.¹ DAG is the endogenous activator of PKC, a serine/threonine kinase that plays an important role in signal transduction.^{2 3} Although a central role for PKC in contraction has been proposed, much of the evidence is contradictory. Protein phosphorylation patterns consistent with PKC activation have been observed in bovine tracheal smooth muscle stimulated with phorbol esters or vasoconstrictor hormones.⁴ However, the identity of the substrate proteins involved in the contractile response is not yet established.⁵ There is evidence in permeabilized vascular smooth muscle cells⁶ and rabbit mesenteric arteries⁷ that PKC activation may be involved in the increased myofilamental Ca²⁺ sensitivity that occurs during tonic contraction possibly through the inhibition of myosin light chain phosphatase via an agonist-mediated G protein,⁸ an effect mediated by arachidonic acid in a PKC-dependent manner.^{9 10} Equally important may be the involvement of the actin-binding protein caldesmon, which is phosphorylated in a PKC-dependent as well as a mitogen-activated protein kinase–dependent manner in arterial smooth muscle cell preparations.^{11 12 13 14} Calponin, another actin-binding protein, may also be involved, because phenylephrine-induced contraction in vascular smooth muscle cells was preceded by a relocation of calponin from cytosol to the surface cortex in a PKC-dependent manner.¹⁵ These data, derived in cultured cells, may provide a link between activation of PKC and sustained contraction. However, whether PKC plays an essential role in agonist-induced tonic contraction in intact smooth muscle preparations is less certain. Haller et al¹⁶ have demonstrated agonist-induced translocation of the enzyme from the cytosol to the particulate fraction in intact bovine carotid artery strips, but the time course did not correlate with the contractile profile for all agonists tested, although this may reflect the likelihood that the agonists used in the above study may use different signaling pathways. Ollerenshaw et al¹⁷ were unable to detect increased phosphorylation of a PKC substrate protein, suggesting nonactivation of PKC in noradrenaline-stimulated rat small arteries. In addition, we and others^{18 19} have been unable to demonstrate a sustained increase in DAG during stimulation with agonists that cause sustained contraction. Conversely, a substantial link between contraction and PKC translocation in response to ₁-adrenergic activation has been documented in dispersed smooth muscle cells.^{6 20 21 22} Phorbol esters, irreversible activators of PKC, and cell-permeant synthetic diglycerides induce a slow and sustained contraction and increase Ca²⁺ sensitivity in intact and permeabilized smooth muscle preparations.^{23 24 25 26} Agonist-induced Ca²⁺ sensitization in rat aorta may be PKC dependent and independent.²⁷ The use of intact vessels in the present study ensures that the architecture of the tissue remains unaltered, such that the system resembles the physiological state; ie, smooth muscle cells are in contact with other cells and are nonproliferative.

PKC comprises a family of several isoenzymes with distinct biochemical characteristics, differential tissue expression, and cellular localization. They all share similar domain structures and are subdivided into three groups: classic PKCs (, ß_I, ß_II, and ), which require Ca²⁺, DAG, and phosphatidylserine for activation; novel PKCs (, , , and ), which have no requirement for Ca²⁺; and atypical PKCs ( and ), which are activated by phosphatidylserine alone.^{2 3} In a variety of cells studied, evidence suggests that the bulk of PKC is present in the cytoplasm, with a rapid redistribution to the particulate fraction after activation, a concept supported by Western blot analysis and immunofluorescence studies.^{28 29 30} These redistribution events appear to correlate with the time course of IP₃ and DAG production by agonist-induced phosphoinositide–phospholipase C activation.^{31 32} IP₃ releases Ca²⁺ from internal stores, which in conjunction with DAG act as the physiological activators of PKC. In addition, hydrolysis of other phospholipids (eg, phosphatidylcholine) also produces DAG but at a relatively later phase in cellular responses.³³ Such observations have led to a general model for coupling of agonists and cellular responses, in which mobilized intracellular mediators induce PKC translocation to a membrane that provides the necessary phospholipids to fully activate PKC. Given that the PKC isoenzymes differ in distribution, regulation, and enzymatic activity, it is likely that individual isoforms may be involved in specific responses. In the present study, we used antibodies to eight of the known isoforms to identify the PKC isoforms present and prolonged phorbol ester treatment to downregulate PKC to investigate the involvement of PKC in agonist-induced contraction of intact rat mesenteric small arteries.

	Materials and Methods

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Tissue Preparation
Female Sprague-Dawley rats (body weight, 200 g) were used for all experiments. Small arteries (internal diameter, <250 µm) from the small intestine were dissected out as already described.³⁴ Vessels were placed in tissue culture medium M199 containing (major constituents only) the following (mmol/L): NaCl 128, KCl 5.4, MgSO₄·7H₂O 0.34, CaCl₂ · 2H₂O 1.3, NaHCO₃ 4.2, KH₂PO₄ 0.44, Fe(NO₃)₃ · 9H₂O 0.002, glucose 5.6, and HEPES 25, equilibrated for 45 minutes at 37°C before all subsequent manipulations.

Tissue Extracts for Western Blotting
Rat mesenteric small arteries were homogenized manually in ice-cold homogenization buffer (20 mmol/L Tris-HCl, pH 7.5, 0.25 mol/L sucrose, 5 mmol/L EDTA, 5 mmol/L EGTA, and 10 mmol/L dithiothreitol) in the presence of 1 mmol/L phenylmethylsulfonyl fluoride and 50 µg/mL leupeptin as protease inhibitors. To identify the PKC isoforms present, SDS sample buffer (diluted 1:5 [vol/vol])³⁵ was added to total homogenates. To determine the distribution of the isoforms, total homogenates were centrifuged at 180 000g for 10 minutes. The supernatant was used as the soluble fraction, and the pellet was resuspended in homogenization buffer as the particulate fraction. SDS sample buffer (diluted 1:5 [vol/vol]) was added to both fractions. All samples were boiled for 5 minutes and either used immediately or stored frozen at -20°C. At each stage, an aliquot was removed for protein estimation using the Bradford assay.

Tissue extracts were subjected to SDS-PAGE on 10% polyacrylamide gels according to the method of Laemmli.³⁵ The resolved proteins were electrophoretically transferred to PVDF membrane by the method of Towbin et al.³⁶ Membranes were blocked in 5% nonfat milk/0.1% Tween-TBS and incubated with the appropriate isoform-specific primary antibody, and signals were developed by horseradish peroxidase–conjugated secondary antibody and an enhanced chemiluminescence detection kit according to the manufacturer's instructions. Signal specificity was demonstrated in parallel blots by competing off antibodies with isoform-specific immunizing peptides.

Downregulation Studies
PKC was downregulated by incubating vessels for 15 hours at 37°C in M199 in a CO₂ incubator with PdBu (500 nmol/L), 4 PdD (500 nmol/L), an inactive phorbol ester, or DMSO (0.1%) alone as vehicle. At the end of the incubations, tissues were washed in ice-cold homogenization buffer to remove residual phorbol esters and processed for Western blot analysis as detailed above.

Functional Studies
After phorbol ester treatment to downregulate PKC, segments of small vessels 2 mm in length were mounted as a ring preparation on wires in a myograph, maintained in physiological salt solution at 37°C for 1 hour, and then set to an internal circumference at which they were held just under tension.³⁷ Cumulative dose-response curves to noradrenaline, AVP (receptor mediated), and KCl+prazosin (10 µmol/L) (non–receptor- mediated) were generated. PdBu (2 µmol/L) was used as a single dose that elicited maximal tension.

Materials
All materials were purchased from Sigma Chemical Co, except for M199 (GIBCO-BRL) and the Western blotting kit (Amersham). Isoform-specific antibodies were raised and characterized as already described.³⁸ Phorbol esters were dissolved in DMSO; vasoconstrictor agonists, in water. All stock solutions were kept at -20°C. Protein concentrations were determined using the BioRad protein assay kit.

	Results

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Expression and Subcellular Distribution of PKC Isoforms in Rat Mesenteric Small Arteries
We used polyclonal antibodies generated against synthetic peptides derived from unique sequences of individual PKC isoforms (, ß₁, ß₁₁, , , , and ) to identify the isoforms present in rat mesenteric small arteries (Fig 1). It is clear from the immunoreactivity with the specific antisera that PKC, PKC, PKC, PKC, and PKC are present. Specificity, as seen by the loss of immunoreactive signal, was demonstrated by the inclusion of isoform-specific synthetic peptide during the primary antibody incubation stage. The inclusion of molecular weight markers as well as PKC from brain indicates small artery isoforms , , and as having apparent molecular masses similar to those present in brain (72 to 90 kD). PKC consistently showed a band of 70 kD instead of 80 kD (brain), whereas PKC showed immunoreactive signals of 75 kD, although occasionally 75, 50, and 30 kD, all of which could be competed off with the inclusion of -specific synthetic peptide.

View larger version (9K): [in this window] [in a new window]   Figure 1. Identification of PKC isoforms in rat mesenteric small arteries. Whole vessels were homogenized, run on SDS-PAGE, electrophoretically transferred to membranes, and probed with isotype-specific primary antibodies as described in "Materials and Methods." Specificity of signal was shown by the absence (lane 1) or presence (lane 2) of isoform-specific synthetic peptide (1 to 5 µg of immunizing peptide). Positions of the molecular mass markers are shown on the right. The figure is representative of results obtained from three experiments.

PKC immunoreactivity in soluble and particulate fractions is presented in Fig 2. It can be seen that PKC was primarily in the soluble fraction (97±1%), whereas PKC demonstrated the converse by being primarily in the particulate fraction (85±3%). PKC, PKC, and PKC differed in their distributions, with amounts in the soluble fractions of 54±5%, 52±3%, and 67±3%, respectively. In agreement with others,³⁸ homogenization of vessels in the presence of Ca²⁺ led to the isoform being localized to the particulate fraction (data not shown). The remaining isoforms did not demonstrate a similar Ca²⁺-induced translocation.

View larger version (24K): [in this window] [in a new window]   Figure 2. The subcellular distribution of PKC isoforms. Extracts were prepared and Western blot analysis was performed as described in "Materials and Methods." Autoradiographs were scanned by laser densitometry, and the results are expressed as a percentage of the total for each PKC isoform. Data are the mean±SEM of seven individual experiments.

Downregulation of PKC and the Contractile Response in Rat Mesenteric Small Arteries
The effect of prolonged incubation with PdBu on PKC isoforms was studied in rat mesenteric small arteries using Western blot analysis of total vessel homogenates. Of the five isoforms present in small arteries, only PKC and PKC were downregulated, as evidenced by a loss of immunoreactive signal. Representative immunoblots are shown in Fig 3. Laser densitometry of autoradiographs from four individual experiments showed an 89±1% loss of PKC and a 92±8% loss of PKC signal intensity. Parallel incubations with the inactive analogue 4 PdD confirmed the presence of the isoforms (Fig 3).

View larger version (13K): [in this window] [in a new window]   Figure 3. The effect of prolonged phorbol ester treatment on rat mesenteric small arteries. A, The contractile response of rat mesenteric small arteries to PdBu (2 µmol/L) was recorded from segments of vessel mounted in a wire myograph after a 15-hour pretreatment with 4 PdD (500 nmol/L, inactive phorbol ester) (i) or PdBu (500 nmol/L) (ii). PdBu (2 µmol/L) was present in the organ bath between administration () and washout (). Tension was recorded continuously. The figure is representative of results obtained from 10 individual experiments. B, Immunoblots are shown of PKC isoforms , , , , and after various 15-hour pretreatments: 1, vehicle (0.1% DMSO); 2, 4 PdD (500 nmol/L); and 3, PdBu (500 nmol/L). The tissues were prepared for immunoblotting as described in "Materials and Methods." The immunoblots are presented to demonstrate the loss of immunoreactive signal only and do not reflect the relative molecular masses of the isoforms, which are shown in Fig 1. The figure is representative of results obtained from a minimum of three experiments.

Similarly, when vessels were incubated overnight with vehicle alone (DMSO), no marked differences in the immunoreactive signal for any of the isoforms were detected between vehicle and 4 PdD treatment when assessed by laser densitometry (minimum of three separate experiments). This suggests that PKC and PKC are indeed being downregulated rather than degraded as a result of overnight incubation. The signal intensities for the , , and isoforms remained unchanged after treatment with PdBu (percent change from 4 PdD: PKC, 2±22%; PKC, 8±8%; and PKC, 0±2% [n=3]).

Overnight incubation with 500 nmol/L PdBu completely abolished the contractile response of the small arteries to phorbol ester, whereas 500 nmol/L 4 PdD pretreatment did not (Fig 3). The dose-response curves and ED₅₀ to noradrenaline and AVP were not different between vessels that had been incubated with PdBu or 4 PdD (Table, Fig 4). There was a significant reduction in the tension developed to submaximal doses of noradrenaline (0.3 and 1 µmol/L) after overnight incubation with PdBu compared with DMSO control (Fig 4). There was no significant difference in the maximal tension developed in response to noradrenaline (Table). The ED₅₀ for KCl-induced contraction was slightly increased after both phorbol ester treatments compared with DMSO control (Table, Fig 4), demonstrating a slight reduction in sensitivity to a non–receptor-mediated stimulus. The maximal tension induced by KCl was not affected by any of the pretreatments (Table). These results suggest that PKC and PKC isoforms are involved in phorbol ester–induced contraction. In contrast, these two isoforms are not essential for noradrenaline-, AVP-, or KCl-induced contraction, although phorbol esters may modulate submaximal noradrenaline-induced contraction.

View this table: [in this window] [in a new window]   Table 1. Contractile Response of Rat Mesenteric Small Arteries to Agonists

View larger version (15K): [in this window] [in a new window]   Figure 4. The effect of increasing concentrations of noradrenaline (A), AVP (B), and KCl+prazosin (10 µmol/L) (C) in mesenteric small arteries after overnight incubation with 0.1% DMSO, 500 nmol/L 4 PdD, or 500 nmol/L PdBu. The data, expressed as percentage of maximal tension developed, are mean±SEM from 10 arterial segments taken from individual animals. *P<.005 for PdBu vs DMSO; +P<.005 for 4 PdD vs DMSO.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Given the importance of small arteries in maintaining flow and tone in the vasculature and the ambiguous role of the PKC pathway, one of the purposes of the present study was to identify the PKC isoforms present in these vessels. Using Western blot analysis with isoform-specific antibodies to PKC, we have identified the isoenzymes present in rat mesenteric small arteries. In agreement with other studies in which multiple isoforms are present in any one system, rat small arteries were no exception. We have identified the following PKC isoforms: and (Ca²⁺ dependent), and (Ca²⁺ independent), and (which can be activated by phosphatidylserine alone). Specificity was demonstrated by blocking immunostaining with coincubation of antibody with immunizing peptide.

That we could detect multiple bands for PKC, all of which could be competed off with synthetic peptide, is not surprising. There is evidence that this isoform is susceptible to rapid proteolysis, because lower molecular weight species have been detected in a variety of cell lines.^{39 40}

The immunoreactive signal for PKC, which could be successfully competed off with the immunizing peptide, was consistently at an apparent molecular mass of 70 kD instead of the expected 80 kD (brain). This may be due to limited proteolysis, despite great care being taken to inhibit any proteolytic activity. Alternatively, the antiserum may be detecting a PKC-related protein.

PKC appears to be restricted primarily to the brain by Western and Northern blot analysis^{41 42 43} and to hippocampal tissue,⁴⁴ with weak immunoreactivity in the adrenal glands,⁴⁵ suggesting that it may be selectively expressed in cells derived from the neural crest. This suggests that the presence of PKC in rat mesenteric small arteries may be from nerve bundles associated with small artery tissue rather than vascular smooth muscle cells.^{46 47}

We determined the subcellular distribution of the individual isoforms. PKC and PKC were mostly in the soluble fraction. PKC and PKC were equally distributed between soluble and particulate fractions, whereas PKC was primarily in the particulate fraction. It should be stressed that our data do not distinguish isoforms that are membrane bound from those associated with the nuclear fraction. Ultimately, the precise spatial distribution of the isoforms as well as the source of the signal can be addressed only by immunocytochemical procedures.

Phorbol esters, which irreversibly bind to and activate PKC, also cause sustained contraction after acute administration. Accordingly, we attempted to identify the PKC isoform(s) involved in phorbol ester–induced contraction. We demonstrated that prolonged treatment with the DAG analogue PdBu resulted in downregulation of only two of the isoforms identified in rat mesenteric small arteries, namely, PKC and PKC. Furthermore, this downregulation was associated with a parallel loss of contractile response to the same agonist, suggesting that PdBu-induced contraction involved PKC and/or PKC only. Although PKC isoforms have been implicated in contractile responses in dispersed/cultured smooth muscle cells,^{20 22} we believe this to be the first instance in which individual PKC isoforms have been specifically identified as being involved in a contractile response in intact vessels, at least when challenged with PdBu. However, downregulation of PKC and PKC did not appear to affect maximal noradrenaline- or AVP-induced contractions, although phorbol esters may reduce the contractile response to noradrenaline at submaximal concentrations. These observations demonstrate quite clearly that the mechanisms involved in phorbol ester– and vasoconstrictor hormone–induced contractions differ.

In rat small arteries, noradrenaline stimulation activates the phosphoinositide signaling system, resulting in elevated IP₃ and intracellular Ca²⁺ with a time course consistent with the initiation of contraction.^{48 49} During the later sustained phase of contraction, small increases in inositol-derived DAG occur.¹⁸ These effects are inhibited by prazosin, which identifies the ₁-adrenergic receptor in this response.¹⁸

Because receptor stimulation by vasoconstrictor hormones generates two intracellular signals that are implicated in PKC activation (ie, production of DAG and raised cellular Ca²⁺ levels),¹ it has been postulated that PKC isoforms may be activated during the contractile response. Consequently, it is of interest to note that of the five isoforms identified in rat mesenteric small artery vascular smooth muscle tissue, three (, , and ) belong to the Ca²⁺-independent group.

Of the two isoforms downregulated by PdBu, only PKC is Ca²⁺ dependent. Given that PKC, the other Ca²⁺-dependent isoform identified in our preparations, is thought to be neuronal in origin, the contractile response elicited by noradrenaline and AVP after downregulation may suggest that if either agonist was using the PKC pathway, they would have to be acting via a Ca²⁺-independent isoform(s). There is some evidence that this may well be the case, at least in ferret aorta, with the ₁-adrenergic receptor agonist phenylephrine. Collins et al⁶ and Khalil et al²² demonstrated phenylephrine-induced contraction of aortic cells at constant Ca²⁺, a response that was blocked by a pseudosubstrate inhibitor peptide of PKC. In parallel, they noted a differential translocation of PKC in a Ca²⁺-independent manner and went on to speculate that at least in their system, the phenylephrine-induced contraction was associated with PKC activation in a Ca²⁺-independent manner. The second isoform present in ferret aorta, PKC, translocated to the intranuclear compartment in agreement with its presumed role in mitogenic signaling.³⁹

Hori et al²⁷ have suggested that two alternate pathways exist for Ca²⁺ sensitization in rat aorta: a PKC-dependent pathway activated by phorbol esters and a PKC-independent pathway activated by receptor agonists. Our results and those of others indicate that this might be a simplistic interpretation. Hori et al have also stated that prolonged phorbol ester treatment of their tissue successfully downregulates all the isoforms present in aorta, an assumption based solely on enzyme activity, which may not be sufficiently sensitive. Indeed, a second study in the rat aorta⁵⁰ has shown that although >95% of PKC activity was lost after 17 hours of treatment with PdBu, there was still a significant contraction to phorbol myristate, suggesting that PKC was still present. Similar to our findings, the contraction to low doses of noradrenaline but not maximal tension was reduced by PdBu pretreatment. In contrast, Marala et al⁵¹ report an attenuation of endothelin-1–induced contraction in porcine coronary arteries after downregulation of PKC by chronic exposure to PdBu, a response reversed by preventing PKC downregulation with 2-chloroadenosine. We have demonstrated by Western blot analysis that of the five isoforms present, only two ( and ) are downregulated. PKC, PKC, and PKC remain unchanged. Indeed, primary sequence information for PKC shows the lack of a typical phorbol ester binding site, an observation that gives this isoform its unique property for not being downregulated.

In conclusion, we have identified the PKC isoforms present in rat mesenteric small arteries, vessels whose primary function is the maintenance of tone in the vasculature. Of the five isoforms present, three belong to the Ca²⁺-independent family. All the isoforms differed in their subcellular distributions, except for PKC, which was primarily localized to the particulate fraction, and PKC, which was mostly soluble, although indirect evidence suggests that the latter might be present in the network of nerves innervating small arteries. The second phase of the present study showed that prolonged treatment of intact vessels with the phorbol ester PdBu resulted in the selective downregulation of only two of the isoforms, with a parallel loss of contractile response to the same agonist. We believe this to be the first report that clearly implicates specific PKC isoforms in phorbol ester–induced intact tissue contraction. Our observations reinforce the view widely held concerning the presence of multiple isoenzymes within the same tissue, namely, that individual isoforms play a role in distinct cellular functions.

	Selected Abbreviations and Acronyms

 

4 PdD = 4-phorbol 12,13-didecanoate AVP = vasopressin DAG = diacylglycerol DMSO = dimethyl sulfoxide IP3 = inositol 1,4,5-tris-phosphate PdBu = phorbol 12,13-dibutyrate PKC = protein kinase C

	Acknowledgments

 
This study was supported in part by the Nuffield and British Heart Foundations. We are grateful to T. Bent for secretarial assistance.

Received November 20, 1995; accepted January 24, 1996.

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