Lack of Cross Talk Between α1-Adrenergic and Angiotensin Type 1 Receptors in Neurons of Spontaneously Hypertensive Rat Brain
Abstract Norepinephrine causes downregulation of angiotensin II (Ang II) receptors in Wistar-Kyoto rat (WKY) brain neuronal cultures. The aim of this study was to compare the cross talk between Ang II and α1-adrenergic receptors in these neuronal cultures. Norepinephrine causes a 66% decrease in Bmax of Ang II type 1 (AT1) receptors in neuronal cultures of WKY brain. This decrease is mediated by the interaction of norepinephrine with the α1A-adrenergic receptor subtype. Norepinephrine also causes a decrease in mRNA levels for AT1 receptors. A maximal decrease of 83% in AT1 receptor mRNA is observed in 8 hours with 100 μmol/L norepinephrine, is blocked by 5-methyluradipil, and involves inhibition of AT1 receptor transcription. Furthermore, decreases in the AT1 receptor and its mRNA are associated with a significant attenuation of AT1 receptor–mediated stimulation of norepinephrine transporter mRNA in WKY brain neurons. In contrast, norepinephrine does not decrease AT1 receptors or mRNA and has no effect on Ang II stimulation of norepinephrine transporter mRNA in neuronal cultures of spontaneously hypertensive rat brain. Thus, these data show that norepinephrine-mediated downregulation of AT1 receptors is associated with a parallel decrease in AT1 mRNA and Ang II stimulation of norepinephrine transporter mRNA and involves the α1A-adrenergic receptor in neurons of WKY brain. This cross talk between the two receptors is lacking in neurons of spontaneously hypertensive rat brain.
- receptors, angiotensin II
- RNA, messenger
- receptors, adrenergic, alpha
- angiotensin II
It is well known that the brain contains a renin-angiotensin system including both AT1 and AT2 receptor subtypes.1 2 3 4 5 6 Interaction of Ang II with its neuronal AT1 receptor subtype initiates a cascade of cellular events that ultimately leads to such physiological effects of Ang II as BP increase, vasopressin release, sympathetic activation, dipsogenic response, and altered baroreflex function.1 2 3 4 5 6 The pressor effect of Ang II partly involves activation of sympathetic nerve activity and is associated with an interaction of AT1 receptors with brain catecholamines. Evidence for this Ang II–catecholamine interaction includes the following: (1) Injection of pressor doses of Ang II into the brain stimulates the synthesis, turnover, and release of norepinephrine from hypothalamic and brain stem nuclei.7 8 9 This action is abolished by antagonists of norepinephrine, dopamine, and α-adrenergic receptors.10 11 12 In addition, catecholamines regulate brain Ang II receptors in a negative feedback fashion.13 (2) Hypothalamic and brain stem nuclei that are involved in the Ang II–induced pressor response also contain catecholamine input/fiber tracts and Ang II and AT1 receptors.14 15 16 17 18 (3) Ang II receptors are present on norepinephrine neurons.19 The relevance of this Ang II–catecholamine interaction in the central control of BP is further supported by studies with SHR, a genetic animal model of hypertension that parallels human essential hypertension.20 These studies demonstrate that Ang II–mediated sympathetic activity is significantly heightened in SHR as a consequence of a hyperactive brain Ang II system.3 4 21 22 Thus, one could conclude from these in vivo studies that the Ang II–catecholamine interaction is pivotal in Ang II–mediated central control of BP and that elucidation of the mechanism of this interaction would be of great significance in furthering our understanding of the pathophysiology of hypertension.
Our group has been studying the cellular and molecular mechanisms of Ang II–catecholamine interactions with the use of an in vitro hypothalamus–brain stem neuronal coculture system from 1-day-old WKY and SHR. These studies have established that a negative feedback interaction exists between Ang II receptors and the catecholamine system that controls the cellular actions of Ang II in neurons. For example, interaction of Ang II with its neuronal AT1 receptor initiates a cascade of cellular events that includes stimulation of the phospholipase C–phosphoinositide–protein kinase C signal transduction pathway and results in stimulation of the NET system and norepinephrine synthesis and release.4 13 The released norepinephrine interacts with the α1-adrenergic receptors, and their chronic activation results in the downregulation of Ang II receptors, thus turning off the neuromodulatory action of Ang II.13 Our objectives in this study were to determine whether (1) downregulation of Ang II receptors by norepinephrine involves the AT1 receptor subtype in neuronal cultures of WKY brain and (2) norepinephrine has a similar or distinct effect on Ang II receptors in neuronal cultures of SHR brain. Evidence shows that the interaction of norepinephrine with α1A-adrenergic receptors downregulates AT1 receptor numbers and mRNA in WKY brain neurons, an action lacking in SHR brain neurons.
One-day-old WKY and SHR were obtained from our breeding colony, which originated from Harlan Sprague Dawley, Indianapolis, Ind. Dulbecco’s modified Eagle’s medium (DMEM), plasma-derived horse serum (PDHS), and crystallized trypsin were from Central Biomedia. Norepinephrine and yohimbine were from Sigma Chemical Co. [α-32P]dCTP (3000 Ci/mmol) was from DuPont-NEN. PCR kits containing Taq DNA polymerase were purchased from Perkin-Elmer Cetus. Reverse transcriptase and a deoxynucleotide mixture containing dATP, dTTP, dGTP, and dCTP were from Stratagene. Oligo(dT)15 was from Promega. RNase inhibitor was from 5′-3′ Prime, Inc. Losartan potassium (formally DuP 753) was a gift from Dr A.T. Chiu of DuPont/Merck. 5-Methylurapidil and chloroethylelonidine were from RBI. Propranolol was purchased from Calbiochem. 125I–[Sar1,Ile8]Ang II (specific activity, 2200 Ci/mmol) was obtained from Dr Robert Speth, Washington State University (Pullman). All other biochemicals were from Fisher Scientific and were of molecular biology grade.
PCR primers for AT1 and AT2 receptors, NET, and β-actin were synthesized by the DNA Synthesis Core Facility of the Interdisciplinary Center for Biotechnology Research, University of Florida. Sequences for these primers were as follows: AT1 receptor: sense, 5′-TTTCTTCTCAATCTCGCCTTGGCTG-3′; antisense, 5′-GGGGATCCAGAAAGCCGTAGAACAG-3′; NET: sense, 5′-CCGCATCCATGCTTCTGGCGCGGATGAA-3′; antisense, 5′-GGGCAGGCTCAGATGGCCAGCCAGTGTT-3′; β-actin: sense, 5′-GAGAAGATGACCCAGATCATGT-3′; antisense, 5′-ACTCCATGCCCAGGAAGGAAG-3′; and AT2 receptor: sense, 5′-GGGAGTCTCTGACAGTTCAAT-3′; antisense, 5′-CCATTGCTAGGCTGATTACAT-3′.
Preparation of Neuronal Cultures From WKY and SHR Brains
Hypothalamus–brain stem areas of brains from 1-day-old WKY and SHR were dissected and brain cells dissociated by trypsin. The hypothalamic block contained the paraventricular nucleus and the supraoptic, anterior, lateral, posterior, dorsomedial, and ventromedial nuclei. The brain stem block contained medulla oblongata and pons. Dissociated brain cells were plated in poly-l-lysine–precoated tissue culture dishes (3×106 cells per 35-mm-diameter dish) in DMEM containing 10% PDHS, and neuronal culture was established essentially as described previously.4 23 24 The cultures were allowed to grow for 10 to 15 days before their use in experiments. These neuronal cultures from both rat strains are comparable in terms of the number of neurons, total cellular protein per dish, and many other biochemical criteria and have been extensively used as an in vitro model in our laboratory for investigation of the cellular and molecular bases of a hyperactive brain Ang II system in SHR.4 Immunohistochemical analysis repeatedly has indicated that the cultures from both rat strains contain 85% to 90% neuronal cells and 10% to 15% astroglial cells.
125I–[Sar1,Ile8]Ang II Binding Assay
Neuronal cultures established in 35-mm culture dishes were incubated with indicated 100 μmol/L norepinephrine for 16 hours in the presence of 10 μmol/L sodium ascorbate. Control cultures contained 10 μmol/L sodium ascorbate essentially as described previously.24 25 26 AT1 receptor binding to untreated and norepinephrine-treated intact neuronal cells attached to culture dishes was determined with the use of 125I–[Sar1,Ile8]Ang II as follows: Growth media were aspirated from cultures, and cells were washed twice with ice-cold PBS (pH 7.4) and incubated with a 0.5-mL reaction mixture containing 1.0 μmol/L 125I–[Sar1,Ile8]Ang II, 1.0% bovine serum albumin, and 1 μmol/L PD 123319 in the absence or presence of increasing concentrations of losartan (0.1 nmol/L to 10 μmol/L).24 After incubation for 1 hour at room temperature, the reaction mixture was removed. Cells were washed twice with ice-cold PBS (pH 7.4) and then dissolved in 0.1N NaOH (0.5 mL per dish). Dissolved cells were transferred into 12/75-mm tubes, and radioactivity was measured in a gamma counter (DP5500, Beckman Instruments). Specific binding, expressed as femtomoles per milligram protein, was calculated by subtracting 125I–[Sar1,Ile8]Ang II bound to cells in the presence of losartan from that bound in its absence. Triplicate culture dishes for total and nonspecific binding were used, and the experiment was repeated three times. Scatchard analysis was carried out from competition-inhibition experiments for the calculation of Kd and Bmax values with the EBDA-ligand program (Elsevier-Biosoft).
Measurements of AT1 Receptor, AT2 Receptor, NET, and β-Actin mRNA Levels
Isolation of Poly(A+) RNA From Neuronal Cultures
Neuronal cultures established in 35-mm dishes were rinsed once with ice-cold PBS (pH 7.4) and lysed by 100 μL lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 0.14 mol/L NaCl, 5 mmol/L KCl, and 1% NP-40). After centrifugation, 100 μL of supernatant was moved to sterile RNase-free tubes containing 3 μL of Dynabeads Oligo(dT)25 (Dynal Inc) and 100 μL of 2× binding buffer (20 mmol/L Tris-HCl, pH 7.5, 1.0 mol/L LiCl, 2 mmol/L EDTA). mRNAs were allowed to bind with Dynabeads for 5 minutes at room temperature, and the poly(A+)-Dynabeads complex was washed and isolated as described in the protocol provided by the company and described by us previously.27
The poly(A+) RNA–Dynabeads complex was suspended in 20 μL RT solution containing 50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L dithiothreitol, 200 ng oligo(dT)15, 200 μmol/L of each dNTP, and 40 IU Moloney murine leukemia virus reverse transcriptase, and the reaction was run for 60 minutes at 37°C. This was followed by heating the reaction for 5 minutes at 65°C. Five microliters of this RT solution was subjected to PCR for the AT1 or AT2 receptor or NET. Two microliters of RT solution was used for β-actin PCR. AT1 and AT2 receptor PCRs in a total volume of 50 μL contained 10 mmol/L Tris-HCl, pH 9.0, 50 mmol/L KCl, 2.5 mmol/L MgCl2, 50 μmol/L of each dNTP, 20 pmol/L of sense and antisense primers, 2 IU Taq DNA polymerase, and 0.1 μCi [32P]dCTP. PCR was performed at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute for 28 cycles. NET PCR and β-actin PCR were performed essentially as described by us previously.27 All these PCR conditions were determined quantitatively for their mRNAs by cyclic lineage analysis described previously.27 β-Actin was used as a standard marker for normalizing the measurements of AT1, AT2, and NET PCR products. PCR products were analyzed on a nondenaturing polyacrylamide gel as described below.
Analysis of [32P]dCTP-Incorporated RT-PCR Products by Nondenaturing Polyacrylamide Gel Electrophoresis
After PCR, 5 μL of the sample containing 32P-labeled PCR products was mixed with 5 μL of 2× gel loading buffer (4% Ficoll 400, 20 mmol/L EDTA, pH 8.0, 0.05% bromophenol blue, and 0.05% xylene cyanole FF) and applied to a 5% acrylamide gel (acrylamide/bis-acrylamide, 29:1) prepared in 1× TBE buffer (89 mmol/L Tris base, 89 mmol/L boric acid, and 2 mmol/L EDTA, pH 8.0). The gel was run for 1 hour at 100 V in a Bio-Rad Mini-Gel System in 1× TBE buffer. The gel was decasted, wrapped in a plastic bag, and exposed to x-ray film overnight at −70°C before development. Each experimental data point was quantified for AT1 receptor, NET, and β-actin mRNAs. Autoradiograms from three independent experiments were used for analysis after normalization with β-actin.
Measurement of PCR Bands
The bands representing PCR products from the x-ray film were scanned with the use of a UVP Imagestore 5000 system (Ultra Violet Products Ltd). The images of PCR bands on X-ray film were captured, and the same level of background and size of scanning area were applied to each individual band. The density of each PCR band was then analyzed and measured with the personal computer software SW 5000 Gel Analysis, and the data were presented as OD units essentially as described previously.25 27 28 The OD units were presented as a ratio of the OD of AT1 receptor, NET, or AT2 receptor mRNA products to the OD for β-actin mRNA product.
Nuclear Run-on Assay
The transcription rate of AT1 receptor mRNA in untreated and norepinephrine-treated neuronal cultures was determined essentially as we described previously.29 In brief, neuronal cultures were established in 100-mm-diameter tissue culture dishes and treated with 100 μmol/L norepinephrine for 16 hours at 37°C. Control cultures incubated with vehicle were used in parallel incubations. Cells (2×107 cells per dish) were scraped and pelleted by centrifugation at 500g for 5 minutes. Nuclei were isolated, the RNA was labeled with [α-32P]UTP, and labeled RNA (6×106 cpm/mL) was hybridized with 5 μg AT1 receptor cDNA probe applied on a nitrocellulose membrane. Radiolabeled bands were measured as described above.
Measurement of AT1 Receptor mRNA In Vivo
Adult male WKY (mean BP, approximately 100 mm Hg) and SHR (mean BP, approximately 190 mm Hg) were housed singly in stainless steel cages in a standard vivarium with Purina Chow pellets and tap water. They were fitted with an indwelling cannula (10 mm long, 23-gauge stainless steel) stereotaxically aimed to end in or just above the lumen of the right lateral ventricle and firmly fixed to the skull with jeweler’s screws and dental acrylic. Surgery was performed with ketamine and xylazine (50 and 5 mg/kg IP) anesthesia, and rats were allowed to recover for 1 week before use in the experiments. Injections were made through an 11-mm, 30-gauge injector needle attached to a 35-mm syringe. Five microliters of either PBS or PBS containing 10 μg norepinephrine was injected into each rat. Twenty-four hours later, rats were killed, and hypothalami were dissected and homogenized in 4 mol/L guanidinium isothiocyanate, 0.01% β-mercaptoethanol, 25 mmol/L sodium acetate, pH 6.0, and 0.5% sacrosyl. Total RNA from each sample was subjected to poly(A+) RNA isolation and AT1 receptor RT-PCR as described for neuronal cultures.
Experimental Groups and Data Analysis
For 125I–[Sar1,Ile8]Ang II binding, each data point was obtained from three 35-mm dishes and each experiment was repeated three times with cells from different rat litters. Similar experimental protocols were used for AT1 receptor, AT2 receptor, and NET mRNA determinations. Data presented are mean±SE and normalized for β-actin mRNA, which was quantified from the same samples for equal loading. Comparisons between experimental and control data were made with one-way ANOVA and Dunnett’s test with the use of Statistica software.
Our first objective was to determine whether norepinephrine-induced downregulation of Ang II receptors observed previously26 30 is a result of its action on the AT1 receptor subtype. Incubation of WKY brain neuronal cultures with norepinephrine caused a dose-dependent decrease in the binding of 125I–[Sar1,Ile8]Ang II to AT1 receptors, with a maximal decrease of approximately 70% that was observed with 100 μmol/L norepinephrine in 16 hours. This decrease in the binding was associated with a 66% decrease in the Bmax for AT1 receptors without significantly affecting the Kd for the binding (Table⇓). In contrast, norepinephrine failed to exert any effect on 125I–[Sar1,Ile8]Ang II binding to AT1 receptors in neuronal cultures of SHR brain.
RT-PCR was used for determination of whether the norepinephrine-induced downregulation of AT1 receptors in WKY brain neurons and the lack of its effect in SHR brain neurons are reflected at the level of AT1 receptor mRNA. The validity of the RT-PCR technique was first established for the measurement of AT1 receptor mRNA in neuronal cultures. Fig 1⇓ shows a linear relationship between the PCR products for AT1 receptor and β-actin in both untreated and norepinephrine-treated WKY brain neurons as a function of PCR cycles. The products were measured by a UVP Imagestore 5000 system, and data are presented as the density of each band. In addition, the data also show that norepinephrine caused a decrease in the AT1 receptor PCR product that was also linear with the PCR cycles. In contrast, norepinephrine had no effect on the β-actin PCR product. An alinear relationship between the PCR products for AT1 receptor and β-actin and the volume of the RT reaction used for the PCR was also observed (Fig 2⇓). This indicated that excellent reproducibility without a significant variability caused by multiple sampling as well as other experimental conditions in either RT or PCR were inherent in this RT-PCR technique. Finally, a linear relationship between the PCR cycles and PCR products was also observed when both AT1 receptor and β-actin reactions were carried out in a single tube (Fig 3⇓). Thus, we concluded from these data that the RT-PCR conditions established for measurement of the AT1 receptor are reflective of its mRNA levels in control and norepinephrine-treated neuronal cultures. Therefore, subsequent experiments were carried out under linear conditions for 28 PCR cycles, and the AT1 receptor mRNA data were normalized by the levels of β-actin PCR products.
Incubation of neuronal cultures of WKY brain with 100 μmol/L norepinephrine caused a time-dependent decrease in AT1 receptor mRNA levels (Fig 4⇓). A maximal decrease of 80% was observed within 8 hours. Basal AT1 receptor mRNA levels were fivefold higher in SHR brain neurons when compared with WKY brain neurons (Fig 4⇓). The decrease in the AT1 receptor mRNA in WKY brain neurons also depended on norepinephrine dose, and a maximal decrease of 83% was observed with 100 μmol/L norepinephrine in 16 hours (Fig 5⇓). The decrease was blocked by 5-methylurapidil and not by chloroethylelonidine, yohimbine, or propranolol (Fig 6⇓), indicating the involvement of the α1A-adrenergic receptor subtype. The norepinephrine-induced decrease in the AT1 receptor mRNA was specific for the AT1 receptor as norepinephrine failed to influence mRNA levels for the AT2 receptor (Fig 7⇓). We carried out nuclear run-on assays to determine the effect of norepinephrine on the transcription rate of AT1 receptor mRNA in WKY brain neurons. Fig 8⇓ shows that the transcription rate of the AT1 receptor was significantly higher in SHR brain neurons compared with WKY brain neurons. This confirms our previous observation that the increased AT1 receptors in this strain are a result of an increase in AT1 receptor gene transcription.4 Fig 8⇓ also shows that norepinephrine caused a 70% decrease in the transcription rate in WKY brain neurons without any influence in SHR brain neurons.
We determined the ability of Ang II to stimulate NET mRNA in untreated and norepinephrine-treated WKY brain neuronal cultures to evaluate whether downregulation of AT1 receptor was associated with a parallel decrease in Ang II responsiveness. Fig 9⇓ shows that Ang II caused a fourfold and sixfold stimulation of NET mRNA levels in neuronal cultures of WKY and SHR brains, respectively. Preincubation of WKY brain neurons with 100 μmol/L norepinephrine for 16 hours resulted in attenuation of Ang II stimulation of NET mRNA levels as there was no significant difference between the mRNA levels in the control and norepinephrine-treated WKY. In addition, norepinephrine failed to have any effect on the ability of Ang II to stimulate NET mRNA in SHR brain neurons.
Finally, we carried out in vivo experiments to validate our in vitro observation of a decrease in AT1 receptor mRNA levels in WKY brain neurons with norepinephrine by measuring the levels of AT1 receptor mRNA in the hypothalami of untreated and norepinephrine-treated WKY and SHR brains. AT1 receptor mRNA levels in the hypothalamus of SHR were threefold higher compared with levels in WKY brain hypothalamus (Fig 10⇓). In addition, 24 hours after norepinephrine injection, AT1 receptor mRNA levels decreased by 56% in WKY brain hypothalamus. In contrast, norepinephrine failed to influence AT1 receptor mRNA levels in the SHR hypothalamus (Fig 10⇓).
Observations from this study demonstrate that previously reported negative feedback regulation of Ang II receptors by norepinephrine13 26 30 is a result of a decrease in AT1 receptor gene expression in WKY brain neurons. In addition, neurons of SHR brain lack this downregulatory mechanism. The action of norepinephrine on AT1 receptors involves the α1A-adrenergic receptor and thus provides an example of the cross talk between these two receptors in the neurons.
The decrease in AT1 receptor mRNA in neurons of WKY brain appears to be due to the effect of norepinephrine on AT1 receptor gene transcription, as evidenced by nuclear run-on assay, with no significant effect on the rate of AT1 receptor mRNA degradation. Although our data are not conclusive as to whether this decrease in the mRNA precedes the action of norepinephrine on AT1 receptor numbers, the relatively slow turnover rate for neuronal receptors13 and the influence of norepinephrine on AT1 receptor transcription argue in its favor. However, other possibilities, such as independent effects of norepinephrine on the receptor and its mRNA, cannot be ruled out at the present time. An interesting aspect of these observations is that they provide a cellular basis for the regulation of Ang II actions in neurons of WKY brain. We propose the following sequence of events based on these and other data: Ang II interacts with the AT1 receptor and stimulates norepinephrine turnover, synthesis, uptake, and release in neurons.4 13 31 There are two consequences of the released norepinephrine. It would be taken up by the neurons via the specific NET system,7 and it would interact with the α1-adrenergic receptors. The latter would downregulate AT1 receptor and turn off the cellular actions of Ang II. This appears to be the only known mechanism by which neuromodulatory actions of Ang II could be regulated in the brain. Finally, our observation of a lack of an effect of norepinephrine on neurons of SHR brain is of great interest given this paradigm. It would suggest that Ang II would persistently stimulate the norepinephrine system in SHR brain neurons because AT1 receptors are not regulated by norepinephrine. Validation of these in vitro data and hypothesis is provided by our in vivo observation presented in Fig 10⇑. This indicates that the downregulatory action of norepinephrine on AT1 receptor mRNA also occurs in the hypothalamus of the adult WKY and that such a mechanism is lacking in SHR. In addition, these data also confirm our previous observations indicating an increase in the basal levels of AT1 receptor mRNA in SHR brain.28
Three important questions arise from these observations. (1) Would an increase in the basal levels of AT1 receptor mRNA and its expression and a lack of their downregulation by norepinephrine observed in neuronal cultures of SHR brain be observed in the neurons of other genetically hypertensive rats? Recent data demonstrating similar observations with the neurons of stroke-prone SHR brain confirm the validity of our observations in SHR.32 In addition, comparisons of observations in SHR with both WKY and Sprague-Dawley rats as normotensive controls further support this conclusion.4 (2) Is the lack of the response of norepinephrine in SHR brain neurons a result of decreases in α1A-adrenergic receptor subtype or an impairment of the signaling mechanism involved in the cross talk between α1A-adrenergic and AT1 receptors? Our data favor the latter possibility because α1-adrenergic receptor levels are in fact increased and not decreased in SHR neurons.33 34 This increase is associated with a parallel increase in both α1A- and α1B-adrenergic receptors and their mRNAs.35 (3) Is the lack of cross talk the basis for increased AT1 and α1-adrenergic receptors in SHR brain neurons? No direct evidence is yet available to answer this question. α1-Adrenergic receptor–mediated signaling pathways should be compared in the neurons from the two rat strains to derive this information. Also, the possibility that differences in norepinephrine response element(s) in the AT1 receptor gene may exist in neurons from the two strains also should be considered. Finally, the regulation of AT1 receptors by norepinephrine seems to be physiologically relevant in the brain on the basis of in vivo experiments that show that both AT1 and α1-adrenergic receptors are increased in the hypothalamic area of SHR27 and that central injections of norepinephrine cause downregulation of AT1 receptor mRNA in WKY brain hypothalamus.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1, AT2||=||angiotensin type 1, type 2|
|PCR||=||polymerase chain reaction|
|SHR||=||spontaneously hypertensive rat(s)|
This research was supported by grant HL33610 from the National Institutes of Health, Bethesda, Md. Dr Di Lu is a postdoctoral fellow of the American Heart Association, Florida Affiliate. We wish to thank Elizabeth Brown for cell culture preparation, Jennifer Brock for the preparation of the manuscript, and Kevin Fortin for graphics.
- Received November 17, 1995.
- Revision received January 8, 1996.
- Accepted February 21, 1996.
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