Spinal Nicotinic Receptor Expression in Spontaneously Hypertensive Rats
Intrathecal administration of nicotinic agonists previously has been shown to result in exaggerated pressor and heart rate responses as well as greater nociceptive behavior in adult (12-week-old) spontaneously hypertensive rats (SHR) than in age-matched normotensive Wistar-Kyoto rats (WKY). Paradoxical to these augmented responses to nicotinic agonists in SHR, nicotinic receptor number in the spinal cord as measured by cytisine binding sites is lower in adult SHR than normotensive WKY and Sprague-Dawley rats. Using the high-affinity agonist epibatidine, we found similar differences in receptor number between strains in both in vitro ligand binding experiments with spinal cord membranes and in situ autoradiographic analyses. Spinal nicotinic receptor number did not differ in 5-week-old prehypertensive SHR and age-matched WKY; however, receptor numbers were higher in young rats of both strains than in their adult counterparts. Antihypertensive treatment (25 mg/kg per day hydralazine PO) in 6-week-old SHR from 6 to 12 weeks of age markedly reduced the progressive rise in blood pressure yet did not alter nicotinic receptor number compared with untreated rats. Similar treatment of WKY with hydralazine produced a slight fall in blood pressure but no change in receptor number. Thus, normalization of blood pressure by hydralazine in SHR does not result in a return of receptor expression to levels seen in normotensive rats. Higher centrally mediated pressor activity or augmented postcoupling events after neuronal nicotinic receptor stimulation may slowly downregulate expression of spinal nicotinic receptors in this genetically hypertensive rat strain.
The central cholinergic nervous system has been implicated in the pathogenesis of hypertension in several models, including spontaneously hypertensive rats (SHR).1 2 3 4 Central administration of cholinergic agonists leads to increases in arterial pressure and heart rate in both SHR and normotensive rats; however, the responses are augmented in SHR.3 4 5 6 Microinjections of nicotinic agonists directly into the rostral ventrolateral medulla also result in exaggerated pressor responses in adult SHR compared with age-matched normotensive rats.5 Furthermore, inhibition of the cholinergic system in young SHR has been shown to delay the development of hypertension.1 7 The roles of spinal cholinergic mechanisms in blood pressure regulation and genetic hypertension are less well defined. Previously, we demonstrated that intrathecal nicotinic agonists elicited exaggerated pressor, heart rate, and nociceptive responses in adult (12-week-old) SHR compared with normotensive age-matched Wistar-Kyoto rats (WKY).8 Furthermore, we reported that the number of nicotinic receptors in the spinal cord of the genetically hypertensive rat was lower than that found in either WKY controls or Sprague-Dawley rats.8 A similar deficiency in nicotinic receptor number has also been observed in various brain regions in SHR compared with WKY,9 contrasting with the reported higher central cholinergic activity in SHR than WKY.9 10 11
The decreased receptor number in hypertensive rats could be a strain difference or a compensatory response to elevated arterial pressure. In the present study, we compared spinal nicotinic receptor numbers in young (5-week-old) prehypertensive SHR and age-matched WKY to determine whether the altered receptor number in SHR occurs before the development of hypertension. We also compared in vitro binding parameters for the high-affinity agonist epibatidine with in situ autoradiographic analysis of receptors to map the anatomic location in the spinal cord where differences in receptor expression may be evident. To ascertain whether elevated arterial pressure directly induces a compensatory response in nicotinic receptor expression, we examined whether chronic antihypertensive treatment (from prehypertensive to adult), which reduces the progression of hypertension in SHR, would normalize the expression of spinal nicotinic receptors.
Male rats used for spinal nicotinic receptor binding measurements and cardiovascular and behavioral testing were matched for age (12 to 16 weeks old). All rats were bred and maintained (12-hour diurnal cycle) within our breeding colony located at the animal care facility at the University of California, San Diego. Genetically inbred SHR and WKY (from stock originally obtained from Charles River Laboratories, Wilmington, Mass) were derived by brother-sister mating; these are designated as SHRLJ and WKYLJ to indicate breeding in La Jolla. All studies were carried out according to protocols approved by the University of California, San Diego, Institutional Animal Care Committee.
Experimental Protocol for Hydralazine Treatment
Twenty-four 5-week-old SHRLJ were randomly separated into two groups of 12; the groups received tap water or tap water plus hydralazine. Five rats from the tap water group were implanted with femoral artery catheters as described below. After 1 week of hydralazine treatment, 6 rats from the hydralazine group were implanted with femoral artery catheters. The first group of 5 rats was used for control mean arterial pressure (MAP) measurements at 0 and 1 week after treatment. MAP measurements for hydralazine-treated rats (the 6 catheterized rats) were initiated at 2 weeks of treatment. The remaining rats from both groups were catheterized 2 weeks after the treatment was started.
MAP was measured every week at the same time of day for each rat. The numbers in parentheses in Fig 3⇓ indicate the number of rats in which MAP was recorded. In rats in which the arterial catheter was blocked, either the catheter was flushed out or the contralateral artery was cannulated in preparation for subsequent measurement of MAP the following week. We discarded 2 rats from the treated group and 1 from the control group because we were unable to measure their MAP after 6 weeks of treatment. At the end of 6 weeks of treatment, the rats (11 from the tap water–treated and 10 from the hydralazine-treated group) were killed; their spinal cords were harvested and membrane preparations were made as described below.
After the study with SHRLJ, we investigated the effects of oral hydralazine treatment on spinal nicotinic receptor binding parameters in WKYLJ. Six and nine WKYLJ between 6 and 7 weeks of age were assigned for vehicle (tap water) and hydralazine treatment, respectively. On the 37th day of treatment, the femoral arteries in both rat groups were catheterized for MAP measurement. MAP and heart rate were measured in both rat groups after 6 weeks of treatment, and rat spinal cords were harvested as described for SHR. We discarded two WKYLJ from the hydralazine-treated group because we were unable to measure their MAP.
Arterial Blood Pressure and Heart Rate Recording
Arterial pressure and heart rate were recorded according to a procedure previously described.12 Arterial catheters were constructed by heat fusing PE-10 and PE-50 polyethylene tubing; the catheters were filled with saline containing 0.9% heparin. With rats under halothane anesthesia, the PE-10 portion of the arterial catheter was advanced via the femoral artery into the abdominal aorta. The free end of the PE-50 tubing was tunneled under the skin and exteriorized at the nape. The catheter was pressure filled with 0.9% heparin-saline and plugged. Rats were monitored daily after surgery, and arterial pressure was measured 4 days after insertion of the catheter. After 2 weeks, catheters needed to be flushed every 2 to 3 days.
Arterial pressure was measured with a Statham P23Db strain-gauge transducer placed at heart level. The transducer signal was conditioned by a pressure processor amplifier (2400S, Gould Instruments), from which pulsatile pressure and MAP were derived. Heart rate was obtained from an ECG/Biotach amplifier (Gould).
Body weight and water consumption were recorded daily for individual rats. On the basis of this information, hydralazine was added to the drinking water to achieve consumption of 25 mg/kg per day for individual rats. Tap water was given to control rats in similar syringe reservoirs.
Hydralazine was obtained from Sigma Chemical Co. [3H]Cytisine and [3H]epibatidine were obtained from New England Nuclear Research. [3H]Cytisine and [3H]epibatidine had specific activities of 30.5 and 56.2 Ci/mmol, and specified purities of 98% and 99%, respectively.
Spinal Cord Membrane Preparation
Membranes were prepared according to a procedure described previously.13 Briefly, a 5-cm (1 cm from the sacral end) segment of the spinal cord was dissected and placed in a polypropylene tube. Tissue sections were stored at −70°C. Spinal cord sections were homogenized in ice-cold 50 mmol/L Tris-HCl buffer, pH 7.4.13 The homogenate was centrifuged at 48 000g for 10 minutes, the pellet was resuspended in fresh buffer and centrifuged a second time, and the final pellet was resuspended in fresh buffer.
Equilibrium Binding Assays
Equilibrium binding assays were conducted according to procedures described earlier for nicotine and cytisine.13 Briefly, the assay mixture consisted of 400 to 600 μg membrane protein in a final incubation volume of 125 μL. Final concentrations of [3H]cytisine and [3H]epibatidine varied between 0.05 and 10 nmol/L (250 to 50 000 cpm) and between 0.02 and 4 nmol/L (250 to 30 000 cpm), respectively; stock solutions were prepared in assay buffer. [3H]Epibatidine and [3H]cytisine incubations were carried out in a cold room (4°C) on a gentle shaker for 60 minutes. Assays were initiated by the addition of the membrane suspension to the [3H]cytisine or [3H]epibatidine solutions in a polypropylene tube with rapid mixing. The incubations were terminated by dilution with 3 mL ice-cold assay buffer immediately followed by rapid filtration under vacuum through Whatman GF/C filter papers previously equilibrated with 0.5% polyethyleneimine at 4°C. Filters were then rinsed three times with 3 mL ice-cold buffer. Specific binding was determined as the difference in binding between samples containing excess unlabeled l-nicotine (40 μmol/L) and those containing only [3H]cytisine or [3H]epibatidine. Protein was assayed by the bicinchoninic acid (BCA) protein assay.
[3H]Epibatidine Binding Autoradiography in Spinal Cord Sections
Halothane-anesthetized rats were transcardially perfused with ice-cold saline immediately followed by 1% formalin. The spinal cord was isolated by hydraulic extrusion and immediately placed on precooled aluminum foil on dry ice. The spinal cord was cut into 1-cm segments with a precooled razor blade, and the segments were immediately dropped in isopentane containers placed in dry ice. The spinal cord segments were placed vertically on precooled chucks with the aid of Tissue-Tek embedding medium. If not sectioned the same day, the tissues with the embedding medium were wrapped in aluminum foil and kept at −70°C. Cryostat sections (cross section, 16 μm thick) were made at −18°C and placed on Fisherbrand lysine-precoated glass slides.
For autoradiographic localization of nicotinic receptors, tissue sections were incubated for 10 minutes in 50 mmol/L Tris-HCl buffer, pH 7.4, containing (mmol/L) NaCl 120, KCl 5, MgCl2 1, and CaCl2 2. This was followed by a second incubation in binding buffer containing 0.6 nmol/L [3H]epibatidine for 60 minutes at 4°C. After incubation, the slides were dipped for 2 minutes four successive times in ice-cold Tris-HCl buffer followed by two quick dips in ice-cold deionized distilled water. The slides were rapidly dried under a stream of cold air. Nonspecific binding was determined by coincubation of tissue sections with [3H]epibatidine and 40 μmol/L l-nicotine.
Dried slides were dipped in Kodak photo emulsion (NTB-2, diluted 1:1 with water) at 40°C to 45°C. The slides were dried, placed in light-tight boxes wrapped with aluminum foil, and stored at 4°C. After 10 to 12 weeks, the emulsion-dipped slides were developed with Kodak D-19 developer and fixer. Grain distribution was observed under a light microscope with the use of dark-field optics. Alternatively, instead of being dipped in photo emulsion, slides were placed against Amersham [3H]-Hyperfilm (with no protective coating on the emulsion) under light-tight conditions for 8 to 10 weeks. At the end of this period, films were hand processed with Kodak D-19 developer and fixer.
All values presented are mean±SE. Student's t test for unpaired data was used for determination of differences between two groups of treatment. Differences between multiple groups were compared by ANOVA.
[3H]Nicotinic Agonist Binding in Spinal Cord of SHRLJ and WKYLJ
Fig 1A⇓ shows the nicotinic receptor number (Bmax) and dissociation constant (Kd) from membrane preparations of the spinal cords of 5- and 12-week-old SHRLJ and age-matched WKYLJ. The number of [3H]cytisine binding sites was lower in the spinal cord membranes of adult SHRLJ than in age-matched WKYLJ. [3H]Epibatidine also exhibited a lower number of binding sites in spinal cord membranes from adult SHRLJ (Bmax, 23.4 and 24.9 fmol/mg protein in two separate preparations) than in membranes from WKYLJ (Bmax, 33.6 and 30.9 fmol/mg protein in two separate preparations) (Fig 1B⇓). In both rat strains, two classes of binding sites were observed with epibatidine (Table 1⇓).
A lower number of spinal nicotinic receptors in SHRLJ compared with WKYLJ was also identified by autoradiographic localization of [3H]epibatidine in various spinal cord segments (Fig 2A⇓). Efforts to analyze [3H]cytisine binding in the spinal cord sections of SHRLJ and WKYLJ by autoradiography were complicated by lower ratios of signal to background levels. As previously described in homogenates from regional areas of the spinal cord,13 greater binding was observed in the dorsal than in the ventral section of the lumbar spinal cord in both SHRLJ and WKYLJ. In the dorsal lumbar section, specific [3H]epibatidine binding was observed in laminae I, II, and III (Fig 2⇓). Moreover, binding was observed in lateral lamina IV and was evident around the central canal. In the ventral lumbar spinal cord, [3H]epibatidine binding was found mostly in laminae VII and IX. In the thoracic spinal cord, binding was greatest in the dorsal horn (Fig 2A⇓). In addition, binding was localized in the intermediolateral cell column region of the spinal column and adjacent lamina VII. As visualized by autoradiography (Fig 2⇓), [3H]epibatidine binding appeared higher in adult WKYLJ than in age-matched SHRLJ for all the regions described.
In contrast to the differences found in adult rats,8 prehypertensive SHRLJ did not differ in spinal nicotinic receptor number compared with age-matched WKYLJ (Table 1⇑). Kd values of the spinal nicotinic receptor for [3H]cytisine also did not differ between 5-week-old SHRLJ and age-matched WKYLJ (Table 1⇑). We obtained tissues for measurement of binding parameters for 5- and 12-week-old SHRLJ and WKYLJ within the same month to minimize divergence within the rat colonies.
As illustrated in Table 1⇑, comparison of the spinal nicotinic receptor number between young and adult rats of each strain showed that receptor number decreased with age in both rat strains. However, the magnitude of the decrease was greater in SHRLJ, thus resulting in significantly lower numbers of receptors in adult SHRLJ than in age-matched WKYLJ.8
Effect of Chronic Treatment With an Antihypertensive Agent on Spinal Nicotinic Receptors in SHRLJ
To ascertain whether the decreased nicotinic receptor in SHRLJ compared with normotensive WKYLJ is reciprocally associated with the development of hypertension in SHRLJ, we treated young SHRLJ with chronic hydralazine (25 mg/kg PO) for 6 weeks before death and measurement of spinal nicotinic receptor density. Fig 3⇓ shows the effect of hydralazine administration on MAP, heart rate, and body weight in SHRLJ from 6 to 12 weeks of age. As expected, MAP of 6-week-old SHRLJ was in the normotensive range. From 7 to 11 weeks of age, untreated SHRLJ exhibited a steady progression to higher resting MAP. Hydralazine-treated rats did not show any rise in MAP for the first 5 weeks (until 10 weeks of age). MAP of treated rats increased slightly, but significantly, from 10 to 12 weeks of age; however, at 12 weeks of age, MAP remained significantly lower in treated rats than in age-matched untreated SHRLJ (P<.002). Thus, the rise in MAP in hydralazine-treated SHRLJ was substantially inhibited compared with vehicle-treated SHRLJ controls (Fig 3A⇓). Hydralazine treatment did not influence heart rate or body weight gain with age compared with corresponding values in untreated rats (Fig 3B and 3C⇓⇓).
Table 2⇓ shows the [3H]cytisine Bmax and Kd values of the nicotinic receptors in spinal cord membranes of hydralazine-treated and control SHRLJ at the end of the treatment period. No differences in [3H]cytisine Bmax or Kd values were observed between the two rat groups, indicating that chronic normalization of blood pressure in SHRLJ does not influence spinal nicotinic receptor number in these rats.
We also determined the effects of chronic hydralazine treatment on nicotinic receptor binding parameters in the spinal cord in WKYLJ. With the use of an experimental protocol similar to that described for SHRLJ, after 6 weeks of oral hydralazine administration, MAP in the hydralazine-treated WKYLJ (90±2 mm Hg) was significantly lower than in tap water–drinking WKYLJ (108±3, P<.004, Table 2⇑). Similar to SHR, no difference in basal heart rate was observed between hydralazine-treated and vehicle-treated WKYLJ at the end of the treatment period (Table 2⇑). Also, no differences were observed in body weight gain between treated and control rats (Table 2⇑). There were also no differences in [3H]cytisine Bmax or Kd values in spinal cord membranes between the two groups of WKYLJ (Table 2⇑).
Central administration of cholinergic agonists into both the brain and the spinal intrathecal space has been shown to lead to exaggerated pressor responses in SHR compared with normotensive rats.4 6 We reported previously that intrathecal administration of nicotine and cytisine elicits dose-dependent increases in blood pressure and heart rate and results in the manifestation of nociceptive responses in both 12-week-old SHRLJ and age-matched normotensive WKYLJ.8 The cardiovascular and behavioral responses to intrathecal nicotinic agonists were exaggerated in SHRLJ compared with either normotensive WKYLJ or Sprague-Dawley rats.8 In addition, SHRLJ exhibited a decreased propensity to desensitize to the cardiovascular and behavioral responses of cytisine.8 The diminished propensity of the cytisine-elicited irritation response to desensitize suggested that amplification of nicotinic receptor–elicited events may be greater in hypertensive than in normotensive rats. We also demonstrated that augmented pressor, heart rate, and nociceptive responses in SHRLJ were accompanied by a paradoxical decrease in nicotinic receptor density in the spinal cord of SHRLJ compared with that of normotensive rats. Receptor numbers in 12-week-old SHRLJ (13.1±0.1 fmol/mg protein) were considerably lower than in both WKYLJ (17.2±1.2)8 and Sprague-Dawley rats (19.9±0.9).13
These findings were extended in the present study, in which we observed that the lower spinal nicotinic receptor number, measured by [3H]cytisine binding sites, in adult SHRLJ compared with WKYLJ was also evident when [3H]epibatidine was used. Epibatidine is reported to be one of the most potent nicotinic agonists for certain neuronal subtypes of receptor14 15 and in rat brain also shows two classes of binding sites.15 In spinal cord preparations of SHRLJ and WKYLJ, [3H]epibatidine exhibited the highest affinity for spinal nicotinic receptors among the radiolabeled nicotinic agonists, and two distinct classes of binding sites were uncovered (Fig 1B⇑ and Table 1⇑). Moreover, similar to other nicotinic agonists, intrathecal epibatidine elicited marked hypertension, tachycardia, and intense irritation responses in rats.16 The rank order potencies of the agonists in eliciting the physiological responses paralleled the order of the binding affinities of the agonists for spinal nicotinic receptors.
A decreased number of spinal nicotinic receptors in adult SHRLJ compared with WKYLJ was also observed by autoradiographic localization of [3H]epibatidine binding sites in spinal cord sections. The significantly higher affinity and signal-to-noise ratio for [3H]epibatidine binding confer additional utility of this radioligand for autoradiographic studies. Except for the superficial layers of the dorsal horn in both thoracic and lumbar spinal cord, SHRLJ showed an overall decrease in [3H]epibatidine binding in the spinal cord.
In addition to revealing differences in receptor number between SHRLJ and WKYLJ, autoradiographic localization of [3H]epibatidine binding demonstrated that spinal nicotinic receptor number was higher in the dorsal than the ventral sections of the lumbar spinal cord. This supports our previous observations in which a greater number of nicotinic receptors were found in membrane preparations from dorsal lumbar than from ventral lumbar spinal cord.13 Autoradiographic analyses of [3H]epibatidine binding in the thoracic spinal cord distinctly identified specific nicotinic binding sites in the intermediolateral cell column region of the thoracic spinal cord, a finding consistent with the high density of nicotinic binding sites found in membrane preparations obtained from this region. Moreover, this region shows the greatest sensitivity in pressor responses elicited by intrathecal nicotinic agonists.17
The lowered spinal nicotinic receptor number in adult SHRLJ compared with WKYLJ was not evident when prehypertensive SHRLJ and age-matched WKYLJ were compared. It is noteworthy that spinal nicotinic receptor number was significantly greater in young rats of both strains. Nicotinic receptor number decreased with age in both strains, but more so in SHRLJ, yielding the difference in spinal nicotinic receptor number between SHRLJ and WKYLJ. Moreover, systemic arterial pressure also increased with age in both rat strains but with much greater magnitude in the hypertensive than the normotensive rats. Since the decrease in receptor density coincided temporally with an increase in systemic arterial pressure, an inverse association between the increase in blood pressure and expression of nicotinic receptors should be considered. Thus, factors that give rise to the enhanced blood pressure in SHR or the consequences of the blood pressure increase may serve to lower expression of spinal nicotinic receptor in this hypertensive rat strain. To help distinguish these alternatives, we sought to blunt the age-dependent development of hypertension by the use of a peripherally acting vasodilator. Hydralazine elicits its antihypertensive action by direct peripheral vasodilatation18 19 ; hence, a central site of action, as attributed to other antihypertensive agents such as captopril or clonidine,20 21 should be minimal. Although chronic hydralazine treatment maintained blood pressure at near normal levels in SHRLJ to 10 weeks of age, spinal nicotinic receptor expression was nevertheless suppressed, as in untreated control SHRLJ. These data would argue against the possibility that the blood pressure elevation accounts directly for the decrease in spinal nicotinic receptor number in SHRLJ.
Acute peripheral vasodilatation and decreased peripheral vascular resistance are likely to result in compensatory increases in central sympathetic activity and heart rate. It could be argued that the antihypertensive action of hydralazine would have reversed the reduction in nicotinic receptor density observed in SHR but was compensated for by an increase in sympathetic activity associated with hydralazine treatment.22 23 Furthermore, SHR are known to have blunted baroreceptor reflexes.24 25 In prior studies, only a transient increase in generalized sympathetic activity was observed after initiation of chronic hydralazine or minoxidil treatment.26 Sustained sympathetic hyperactivity was noted only for the heart, implying an organ-specific effect that could be attributed in part to volume overload and/or ventricular distention.26 Moreover, these previous studies did not reveal generalized and long-term increases in sympathetic activity, as measured by catecholamine turnover or hexamethonium-elicited reductions in blood pressure.26 Although after 35 days of hydralazine treatment we found that heart rate was not increased in SHR, an indirect effect of hydralazine on central sympathetic outflow cannot be completely excluded. Thus, the effect of chronic hydralazine administration on spinal nicotinic receptor number in WKY was also examined. Since WKY have significantly lower sympathetic activity than SHR,24 the lack of influence of chronic hydralazine treatment on spinal nicotinic receptor numbers in WKY further suggests that a chronic rise in blood pressure does not alter spinal nicotinic receptor in the genetically hypertensive rat model.
Similar to our findings, Yamada et al9 observed decreases in nicotinic receptor number in various brain regions, including the medulla oblongata, of stroke-prone SHR compared with WKY. Microinjections of nicotine in the rostral ventrolateral medulla produced a dose-dependent increase in blood pressure and heart rate in SHR, WKY, and Sprague-Dawley rats.5 The pressor and tachycardic responses to nicotine were augmented in SHR compared with normotensive rats.5 Taken together with the findings reported here, these results show that augmented pressor and heart rate responses to nicotinic receptor stimulation in the presence of a decreased number of nicotinic receptors are observed in both the brain and spinal cord.
Increased cholinergic activity in various brain regions has been demonstrated in SHR compared with WKY,10 11 and inhibition of the cholinergic system by intracerebroventricular hemicholinium-3 led to a fall in blood pressure in hypertensive rats.1 7 Thus, the lower nicotinic receptor numbers in the brain regions of hypertensive versus normotensive rats could be explained by a feedback mechanism whereby SHR partly compensate for the increased cholinergic activity in the brain and spinal cord. In contrast to the situation in brain, hemicholinium-3 administration in the spinal cord did not alter blood pressure in SHR.6 Moreover, intrathecal cytisine administration, in addition to evoking exaggerated cardiovascular and behavioral responses in SHRLJ, elicited greater release of the excitatory amino acids glutamate and aspartate in spinal microdialysates of SHRLJ than in WKYLJ.16 This finding is consistent with the argument that the amplification of the postcoupling events after spinal nicotinic receptor stimulation may be augmented in SHR.
The basis for the reduction of nicotinic receptor number can only be a matter of speculation at this stage of development. Studies with chronic nicotine or N-methylcarbamylcholine treatment demonstrate that these agents can cause an anomalous increase in receptor number.27 28 This may partly be the consequence of nicotine both stimulating and desensitizing receptors, a phenomenon that might not be paralleled by cholinergic stimulation and excitability elicited by released acetylcholine. Excessive sympathetic activity may also be involved in the regulation of nicotinic receptor number, and this could arise either at the spinal or supraspinal level. Projected studies are directed at resolving whether cholinergic or adrenergic activity in higher brain centers or in the spinal cord influences nicotinic receptor expression.
This work was supported by US Public Health Service Grant HL-35018 and a grant from the University of California Tobacco Related Disease Research Program to P.T. We gratefully acknowledge the help of Dr Mark Ellisman for allowing us to use the National Center for Microscopy and Imaging Research (RR04050).
- Received April 22, 1996.
- Revision received May 28, 1996.
- Revision received July 30, 1996.
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