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Endocrinology Vol. 138, No. 7 2658-2664
Copyright © 1997 by The Endocrine Society

ARTICLES

A Novel Calcium-Activated Apamin-Insensitive Potassium Current in Pituitary Gonadotrophs

Leoncio Vergara, Eduardo Rojas and Stanko S. Stojilkovic

Laboratory of Cell Biology and Biochemistry, National Institute of Diabetes and Digestive and Kidney Disorders, and Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Stanko Stojilkovic, NICHD/ERRB/UCS, Building 49, Room 6A-36, 49 Convent Drive, MSC 4510, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In cultured rat pituitary gonadotrophs, GnRH-induced oscillations in cytosolic calcium concentration ([Ca2+]i) are associated with periodic membrane hyperpolarization. The hyperpolarizing waves are secondary to the activation of apamin-sensitive Ca2+-activated K+ channels that account for a single class of 125I-apamin binding sites present in these cells. In a substantial fraction of gonadotrophs, however, we observed a Ca2+-controlled oscillatory current that was resistant to apamin, even at concentrations five orders of magnitude higher than the dissociation constant (Kd) observed in the binding experiments. With the K+ in the pipette, the apamin-resistant current showed a reversal potential of -42 mV, nearly 40 mV more positive than that of the apamin-sensitive current. With Cs+ in place of K+ in the pipette solution, both the size of the apamin-insensitive current and its reversal potential remained unchanged. Ion substitution studies further revealed that the reversal potential was independent of Cl-. In contrast, an 11 mV hyperpolarizing shift in the reversal potential occurred when extracellular Na+ was reduced to 80 mM. In cells expressing apamin-resistant conductances, addition of apamin evoked a marked increase in the duration of the action potentials and reduction in the frequency of spontaneous spiking. In the presence of GnRH, gonadotrophs exhibit the typical burst pattern of electrical activity. Further exposure of the cells to apamin depolarized the membrane from a silent phase bursting level of about -80 mV to a new level of about -40 mV. These observations indicate that, in addition to apamin-sensitive current, a subpopulation of pituitary gonadotrophs also expresses a cationic component of the Ca2+-activated membrane conductance that has the potential to remodulate spontaneous and agonist-induced electrical activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TWO CALCIUM signaling systems are operative in rat pituitary gonadotrophs: voltage-dependent and intracellular calcium mobilization-dependent. A set of plasma membrane channels expressed in pituitary gonadotrophs (1, 2, 3, 4, 5, 6) provides the mechanism for plasma membrane-derived Ca2+ signaling that occurs in the form of spontaneous action potentials (APs) that drive cytosolic calcium ([Ca2+]i) fluctuations (7, 8). Consistent with the role of L-type calcium channels in such signaling, spontaneous firing of APs and the associated fluctuations in [Ca2+]i are abolished by addition of the dihydropyridine calcium channel antagonist, nifedipine, or when the cells are exposed to a Ca2+-deficient medium (7). On the other hand, Ca2+-mobilizing agonists, such as GnRH (9, 10, 11, 12), endothelins (10), and pituitary adenylate cyclase-activating polypeptide (13), initiate [Ca2+]i, oscillations due to InsP3-induced calcium mobilization from intracellular stores. As in other cell types (14), the InsP3-dependent spiking shows frequency modulation in response to increases in GnRH concentration (15). Agonist-induced elevations in [Ca2+]i are synchronous with episodes of membrane hyperpolarization (16). These hyperpolarizing events periodically interrupt the spontaneous electrical activity of gonadotrophs, generating a bursting pattern in the firing of APs (7, 16). Calcium entry driven by AP-induced depolarization of the cells is essential for the sustained agonist-induced and InsP3-dependent Ca2+ spiking in these cells (7, 9).

In general, the coupling factors between voltage-dependent Ca2+ entry and InsP3-dependent Ca2+ release are Ca2+-sensitive currents (17). In gonadotrophs, apamin-sensitive Ca2+-activated K+ channels (SK-KCa) are the predominant mediator of the Ca2+-dependent plasma membrane conductance (12, 16). The SK-KCa channels are also expressed in other pituitary cells types, including corticotrophs (18), lactotrophs (19), thyrotrophs (20), and immortalized GH3 cells (21, 22), as well as in other endocrine cells (23, 24). These channels are voltage-insensitive and show a higher sensitivity to Ca2+ than other Ca2+-activated K+ currents. This renders them appropriate for controlling the interspike interval by producing long hyperpolarizing pauses after APs, as well as spike frequency adaptation during depolarizing pulses (25). Consistent with this, an increase in the AP spiking frequency follows the application of apamin in GH3 pituitary cells (22). Also, addition of apamin to cultured pituitary cells leads to an increase in ACTH, TSH, and PRL release (19, 26). We have recently observed that gonadotrophs express another Ca2+-activated conductance, which is apamin-resistant and present in a substantial fraction of the gonadotroph population. The main objective of the present work was to characterize this Ca2+-sensitive and apamin-resistant current, and to gather information on its selective expression and possible physiological role in a subpopulation of gonadotrophs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Gonadotrophs were isolated from the pituitaries of ovariectomized Sprague-Dawley rats by enzymatic dispersion, and maintained in primary culture, as previously described (7). Briefly, rats were ovariectomized 14 days before each cell culture preparation, and the tissue from four to ten pituitaries was minced and incubated in PBS supplemented with 1.5 mg/ml trypsin (Sigma Chemical Co., St. Louis, MO) and 1 mg/ml DNAse I (Boeheringer Mannheim, Indianapolis, IN) at 37 C for 1 h. After mechanical dispersion of the tissue and washing with fresh PBS, cells were suspended in Medium 199 (Life Technologies, Grand Island, NY), plated in plastic culture dishes (Nunc, Roskilde, Denmark), and kept at 37 C in a 95% air/5% CO2 atmosphere for 2–4 days before recordings were made.

125I-labeled apamin binding
Binding studies on intact cells were performed at 22 C with cultured pituitary cells from normal and ovariectomized rats. Apamin (Sigma) dissolved in binding medium consisting of medium 199 with 25 mM HEPES, pH 7.4. The cells were washed twice with binding medium and then incubated for 90 min in medium containing 10 pM 125I-labeled apamin (Amersham, Arlington Heights, IL) and nonradioactive apamin to be evaluated for competitive binding activity. After incubation to equilibrium, the cells were washed rapidly three times with ice-cold PBS/0.1% BSA, then solubilized in 1 M NaOH containing 0.1% SDS and analyzed for bound radioactivity in a {gamma}-spectrometer.

Electrophysiological recording
Membrane potential and whole cell currents were measured using the nystatin perforated patch-clamp technique (16). If not otherwise specified, the culture medium was replaced with a solution containing (in mM): 140 NaCl, 4 KCl, 2.6 CaCl2, 1 MgCl2, 10 HEPES (hydroxipiperazine ethano sulfonic acid, sodium salt), and 5 glucose. The pH was adjusted to 7.36 using NaOH. Patch-clamp pipettes were made of soft capillary glass (Blue-Tip, Oxford) using a BB-CH-PC puller (Mecanex, Geneva, Switzerland) and had a tip resistance between 2 and 4 M{Omega}. The composition of the pipette solutions are described in the figure legends. Nystatin (Sigma) was added from a stock solution to obtain a final concentration of 100–200 µg/ml. The signals were recorded under voltage and current clamp conditions using an EPC-7 patch clamp amplifier (List, Darmstadt, Ebenstadt, Germany). A 140 mM NaCl-agar bridge was placed between the bathing solution and the reference electrode. For analysis, records were filtered at 50 Hz and digitized at 100 Hz, using the software packages Axotape 2.0 and Pclamp 5.5 from Axon Instruments (Foster City, CA). All reported membrane potentials were corrected for liquid junction potentials, calculated using the JPCalc program (15).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
It is well established that GnRH-induced [Ca2+]i oscillations in pituitary gonadotrophs are associated with activation of Ca2+-sensitive conductancies (7, 12, 16, 27). Figure 1AGo shows a typical pattern of agonist-induced Ca2+-dependent oscillatory current in an isolated gonadotroph. At a holding potential of -64 mV, GnRH induced a transient outward current followed by regular oscillatory outward spikes. Figure 1BGo shows changes in the amplitude and direction of the current in response to a voltage ramp (panel C); the plasma membrane potential (Vm) was changed from -64 to -94 mV and then increased to -34 mV over a 3.5-min period. The initial hyperpolarization changed the direction of the current. As the cell was progressively depolarized, the amplitude of the inward current was reduced and at a Vm of -81 mV the direction of the current changed from inward to outward. The averaged Erev is -83 mV (n = 5), which is similar to the calculated equilibrium potential for K+, which under our experimental conditions was close to -85 mV.



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  		Figure 1. Characterization of GnRH-induced oscillatory current in pituitary gonadotrophs. A, The pattern of GnRH-induced current oscillations in a cell held at -64 mV. B, Alterations in the amplitude and direction of the oscillatory current in response to changes in Vm induced by a ramp protocol (C) from -94 to -34 mV over 3.5 min. The holding potential previous to the ramp was -64 mV. As indicated by the dashed lines, the reversal potential (Erev) for this oscillatory current was -81 mV. In these experiments, as well as those shown in Figs. 2Go and 3Go, the composition of the intrapipette solution was (in mM): KCl 20, K-aspartate 120, MgCl2 3, and NaHEPES 10.

 
In all analyzed cells, apamin reduced the amplitude of GnRH-induced oscillatory current, consistent with the conclusion that all gonadotrophs express SK-KCa. In about 45% of gonadotrophs held at -64 mV, apamin abolished the oscillatory currents (Fig. 2Go, left panel) without affecting the underlying [Ca2+]i oscillations (not shown). This observation indicates that Ca2+-activated oscillatory current in a subpopulation of pituitary gonadotrophs is exclusively conducted through SK-KCa channels. In the residual cells (26 of 47), however, inhibition of the main component by 1 µM apamin unmasked an inward component of the current at -64 mV (Fig. 2Go, right panel). Furthermore, the residual oscillations, recorded in the presence of apamin reverted from inward to outward when Vm was changed from -54 to -34 mV, with an estimated Erev of -42 mV (Fig. 3Go). This reversal potential is 39 mV more depolarized than the potential of the GnRH-induced apamin-sensitive oscillatory current and is far from the Nerst equilibrium potential for K+.



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  		Figure 2. Effects of apamin on oscillatory currents induced by GnRH. In a fraction of cells held at -64 mV, apamin completely inhibited the agonist-induced oscillatory current (left panel). In remaining cells, inhibition of the outward current by apamin unmasked an inwardly oriented oscillatory current (right panel).

 


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  		Figure 3. Effects of Vm on the residual apamin-insensitive oscillatory current induced by 100 nM GnRH. The pattern of changes in Vm is shown in the upper panel, and the corresponding changes in whole-cell current in the bottom panel. As in Fig. 2Go, at -64 mV the oscillations appear as inward deflections, in contrast to the outward deflections normally observed in the absence of apamin. There was a switch in the polarity of the oscillations in response to changes in Vm between -54 and -34 mV, giving an estimated value of -42 mV (n = 10) for Erev.

 
In general, the lack of effects of apamin could be explained by the partial inhibition of SK-KCa. However, in cultured pituitary cells from normal (not shown) and ovariectomized female rats (Fig. 4Go), 125I-apamin binding was inhibited by unlabeled apamin in a monophasic manner, with IC50 s of 25 and 30 pM, respectively. In both cases, Scatchard analysis was consistent with the expression of a single class of typical apamin receptor-channels. The calculated number of channels per cell in six independent experiments ranged between 100 and 250. Thus, it is unlikely that partial inhibition of SK-KCa by apamin accounts for the observed effects.



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  		Figure 4. Competitive inhibition of 125I-apamin binding by unlabeled apamin in cultured pituitary cells from ovariectomized rats. Pituitary cells were incubated with 10 pM 125I-apamin in the absence and presence of unlabeled apamin. Nonspecific binding was estimated in the presence of 10 µM unlabeled apamin. B/Bo is the fraction of 125I-labeled apamin specifically bound in the presence of increasing concentrations of apamin. The dashed line indicates the IC50 value.

 
In experiments with Cs+ as the predominant intracellular cation, the apamin-resistant component of calcium-activated current was also observed in GnRH-stimulated gonadotrophs. The Erev of this oscillatory Ca2+-activated current, estimated by changing the holding potential, was approximately -43 mV (Fig. 5Go), which is inconsistent with either SK-KCa or BK-KCa. Two other possibilities for the Ca2+-activated conductance are Cl- and cationic nonselective channels. When the intrapipette Cl- was reduced from 70 to 10 mM, the reversal potential of the apamin-resistant oscillatory currents was not significantly modified (-45 mV, Fig. 6AGo). Because the nystatin pores are also permeable to monovalent anions (28), this experiment indicate that a Cl- conductance could not explain the apamin-resistant current. In contrast, when the extracellular sodium was reduced from 140 mM to 80 mM, a negative shift of 11 mV in the reversal potential was observed (Fig. 6BGo). This shift was less than expected for a Na+-permeable cationic nonselective conductance (29). Such characteristics of apamin-resistant current in gonadotrophs also differ from that reported for a pure apamin-resistant SK-KCa current in chromaffin cells (23, 24).



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  		Figure 5. Effects of changing the holding potential on GnRH-induced apamin-insensitive current oscillations-I. The recording was taken from a cell bathed in 1 µM apamin and with a pipette solution containing cesium instead of potassium. The upper panel shows the current trace and the lower panel shows the relationship between current and membrane potential. The estimated Erev is at -43 mV. In the experiment illustrated in this figure, the composition of the intrapipette solution was (in mM): CsCl 70; Cs-methanesulfonate 70, MgCl2 3, and Na-HEPES 10.

 


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  		Figure 6. Effects of changing the holding potential on GnRH-induced apamin-insensitive current oscillations-II. Upper panel, Lack of effect of the partial substitution of intrapipette chloride with methanesulfonate on Erev; the current direction reversed at -45 mV, comparable to that observed in the controls. Lower panel, the shift in Vm dependence of apamin-insensitive oscillatory current induced by reduction of sodium in the extracellular medium. The extrapolated value for Erev is -44 mV. The composition of the intrapipette solution in both experiments was (in mM): CsCl 10, Cs-methanesulfonate 130, MgCl2 3, and NaHEPES 10. The extracellular solution was modified by reducing NaCl from 140 mM to 83 mM, the difference being replaced with N-methyl-D-glucamine chloride (lower panel). The recordings were taken from cells bathed in the presence of 1 µM apamin.

 
We have also analyzed the pattern of electrical activity in spontaneously active and GnRH-stimulated gonadotrophs expressing apamin-resistant current. As reported earlier (5, 7, 16), gonadotrophs frequently exhibit spontaneous electrical activity. Addition of apamin (1 µM) in such cells was followed by changes in the duration and frequency of spontaneous APs (Fig. 7AGo). Figure 7BGo, left panel, illustrates the pattern of the electrical activity in controls (C) and after the application of 1 µM apamin (A). Furthermore, blockade of apamin-sensitive current was without effect on both the threshold and depolarization during the APs. However, the repolarization phase of APs was prolonged, and the subsequent afterhyperpolarization was abolished. As shown in Fig. 7BGo, the sustained depolarization was also affected by apamin. The dashed horizontal line indicates that changes in the slow depolarization occurs at a potential close to the Erev for the apamin-resistant current. Table 1Go summarizes the effects of apamin on the frequency and duration of APs in spontaneously active cells, in which the presence of apamin-resistant conductance was subsequently identified by addition of GnRH under a voltage-clamp.



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  		Figure 7. Effects of apamin on spontaneous electrical activity in pituitary gonadotrophs. A, The pattern of electrical activity in a cell before and after the exposure to apamin. B, left panel, effects of apamin on the repolarization phase of APs. The dotted vertical line indicates that the plateau phase of APs coincides with afterhyperpolarization phase in controls. B, right panel, effects of apamin on the sustained depolarization phase. C, control, A, apamin (1 µM)-treated. The dotted horizontal line indicates the Erev value for the resting potential observed under identical ionic conditions (Fig. 3Go). The identification of GnRH-induced apamin-insensitive current was carried out after addition of GnRH.

 

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  		Table 1. Effects of apamin (1 µM) on frequency and duration of action potentials in spontaneously active gonadotrophs

 
The effects of apamin on GnRH-induced electrical activity were also analyzed. In free running gonadotrophs, GnRH induced periodic hyperpolarization of the membrane, followed by periodic bursting. As reported earlier (16), application of apamin leads to abolition of these periodic hyperpolarization events (Fig. 8AGo). Furthermore, after exposure to apamin, the burst pattern of GnRH-induced electrical activity was replaced with a different pattern of activity, in which APs exhibited a slow repolarization and the level of firing was about -40 mV. Two examples of such pattern are shown in Fig. 8Go, B and C. The time-course of the voltage and current oscillations, shown in panel C, are consistent with the idea that activation of the apamin-resistant channels leads to membrane depolarization. As illustrated by the dotted line, the projected voltage spike coincides with initiation of an inward current transient.



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  		Figure 8. Effects of apamin on GnRH-induced electrical activity in pituitary gonadotrophs. A, Abolition of the hyperpolarization phases in GnRH-induced electrical activity. B and C, A plateau-like firing after addition of apamin. C, Illustration that GnRH-induced apamin-insensitive current was present in this cell. The time-courses of voltage and current oscillations are consistent with a depolarizing nature of the apamin-resistant current. The dotted line illustrates the beginning of projected voltage spike.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In female rats, the pituitary gonadotroph population is highly dynamic and exhibits increases during diestrus and after ovariectomy and a substantial decrease after the GnRH surge and during lactation. The augmentation of gonadotroph population is the result of two different processes, mitosis and entry of nonsecretory cells into the active gonadotroph pool (30). It is also associated with an increase in cell size (31), transformation from monohormonal to bihormonal cells (32), and increase in the expression of LH-ß and FSH-ß subunit messenger RNAs (33, 34, 35, 36). These data have led to the conclusion that the gonadotroph population is composed of several subpopulations: resting and active, nonsecretory and secretory, monohormonal and bihormonal, mitotic and recycling, etc. It is reasonable to speculate that the variety of developmental and cycle-dependent stages in gonadotrophs is associated with qualitative and/or quantitative differences in their intracellular signaling properties. Previously, we (15) and others (11, 37) reported the existence of the quantitative differences in GnRH-induced calcium signaling in gonadotrophs from neonatal, prepubertal, peripubertal, cycling, and ovariectomized female rats. Here, we presented evidence for the expression of a novel calcium-dependent and apamin-insensitive cation-selective channels that is coexpressed with apamin-sensitive SK-KCa channels. Because gonadotrophs from ovariectomized rats employed in these studies predominantly originate from new mitotic cells (30), these observations may suggest that the apamin-insensitive component is expressed during their maturation.

The presence of apamin-sensitive and -insensitive components of Ca2+-activated currents is not unique to gonadotrophs. Pituitary corticotrophs (26, 38) and GH3 immortalized lacto-somatotrophs (21, 22, 39) also express apamin-sensitive and -insensitive Ca2+ activated currents. In both cell types, the apamin-insensitive component was carried through BK-KCa, which are sensitive to tetraethylammonium and carybdotoxin. The intrinsic properties of BK-KCa channels are appropriate for regulation of AP duration and the associated Ca2+ entry through voltage-sensitive calcium channels. On the other hand, the SK-KCa channels are suitable for the control of AP frequency due to their role in control of a sustained (slow) afterhyperpolarization (25). Thus, inhibition of these channels would be expected to increase AP frequency, leading to a net increase in Ca2+ entry and secretion (8). In accord with this, apamin has been shown to increase AP frequency in about 30% of GH3 immortalized lacto-somatotrophs by diminishing the slow hyperpolarization between APs (22).

In addition to these channels, chromaffin cells also express a minor apamin-insensitive conductances. Electrophysiological and pharmacological characterization of this residual current is consistent with the presence of another subclass of SK-KCa that are resistant to apamin (23, 24). However, several lines of evidence strongly suggested that apamin-resistant Ca2+-controlled conductance differs from BK-KCa or apamin-insensitive SK-KCa. There is a clear change in the reversal potential of GnRH-induced oscillations observed after addition of apamin. In experiments using Cs+-rich pipette solution, there are no changes in the amplitude of GnRH-induced apamin-resistant current and Erev, which are commonly observed in cells expressing SK-KCa channels (23). Furthermore, our binding data indicate that apamin binds to a single class of channels, with a Kd in the pM concentration range, that is comparable to that found in other tissues (40, 41, 42). This observation argues against the partial inhibition of SK-KCa channels.

In addition to K+ channels, the family of Ca2+-activated channels includes the chloride and nonspecific ionic channels. Ca2+-activated Cl- channels are permeable to several other small anions, and the open probability of these channels is controlled by [Ca2+]i and depolarization (17). It is unlikely that apamin-resistant conductance observed in a subset of gonadotrophs is carried through these channels because a decrease in intrapipette Cl- concentration from 70 to 10 mM and increase in equilibrium potential from -18 mV to -57 mV did not affect the Erev for GnRH-induced oscillatory current. On the other hand, the decrease in extracellullar Na+ concentration induced a negative shift in Erev, suggesting that in addition to K+ and Cs+, apamin-resistant channels also conduct Na+. This shift was less than expected for a typical Ca2+-activated nonselective cationic channels (43), suggesting that apamin-resistant channels belong to the cationic family of channels, with higher selectivity for K+ than Na+.

It is likely that apamin-sensitive and -resistant channels play an important role in the control of spontaneous and agonist-induced electrical activity. In cells that express apamin-resistant component of Ca2+-activated current, addition of apamin induces significant changes in the pattern of spontaneous and agonist-induced electrical activity. In the absence of apamin, a regular discharge of APs was observed in gonadotrophs (7, 16). After addition of apamin, the frequency of APs decreases as a consequence of the prolongation of the sustained afterhyperpolarization and depolarization phases. The duration of spikes also increases due to the delay in the repolarization phase of spikes and abolition of an initial afterhyperpolarization. These observations suggest that apamin-sensitive channels play a more complex role in gonadotrophs than in other cell types (21); in addition to afterhyperpolarization, they also control AP duration. The apamin-resistant channels control the sustained slow hyperpolarization/depolarization phase in these cells. Thus, with the Erev close to the resting potential of these cells, apamin-resistant channels can stabilize the membrane potential. Consistent with these conclusions, no increase in the frequency of spiking was observed in any of cells after addition of apamin, an effect consistently observed in other cells types (reviewed in Ref. 17).

As addressed earlier (16), GnRH-induced cycling hyperpolarization of the membrane are abolished by apamin. Also, the bursting pattern of firing after the hyperpolarization waves was replaced by a plateau potential pattern. Similar patterns of firing were observed in pituitary melanotrophs and were driven by nonselective cationic channels, which activation leads to depolarization of cells (29). In gonadotrophs, the time-courses of voltage and current oscillations in GnRH-stimulated and apamin-treated cells are also consistent with the depolarizing nature of the apamin-resistant current. It is possible that the rise in [Ca2+]i due to calcium mobilization activates these channels, which in turn depolarize the cells at the resting membrane potential. With a blockade of apamin-sensitive channels such depolarization is leading to the plateau type of potential. Thus, the interplay between apamin-sensitive and -resistant channels regulates the pattern of spontaneous and agonist-induced electrical activity in gonadotrophs.

However, in the absence of specific activator and/or inhibitor of apamin-resistant channels, it is difficult to discussed the specific role in regulation of membrane excitability. A detailed analysis of ionic selectivity, voltage and Ca2+ sensitivity of this novel conductance is essential for a better understanding of the observed effects of apamin on the pattern of electrical activity. The finding that only a subset of pituitary gonadotrophs expresses this conductance that is operative in unstimulated and agonist-stimulated cells also raises several endocrinological questions that should be addressed in future experiments. Cytological and cytochemical analysis of gonadotrophs will be required to identify the subset of cells that express these channels. Once available, such data could also help in the purification of these cells, and in the analysis of hormone synthesis and secretion, and gene expression, in this specific subpopulation of gonadotrophs.


   Acknowledgments
 
We thank Manuel Kukuljan and Fredrick Van Goor for their helpful discussions.

Received March 10, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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