Differential Expression of Ionic Channels in Rat Anterior Pituitary Cells

Fredrick Van Goor, Dragoslava Zivadinovic and Stanko S. Stojilkovic

Endocrinology and Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892-4510


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+-sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+-activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The anterior pituitary is composed of the five major hormone-secreting cell types, corticotrophs, lactotrophs, thyrotrophs, somatotrophs, and gonadotrophs. Corticotrophs arise from a lineage that is distinct from the other cell types, whereas the remaining cell types share common transcription factors and frequently produce multiple hormones, indicating that they are closely related (1). Despite their similar origin, somatotrophs, lactotrophs, and gonadotrophs differ with respect to their pattern of spontaneous electrical activity, intracellular Ca2+ ([Ca2+]i) signaling, basal hormone secretion, and neuroendocrine regulation of spontaneous Ca2+ influx and Ca2+-dependent hormone secretion. Specifically, somatotrophs and lactotrophs exhibit extracellular Ca2+-dependent, high-amplitude [Ca2+]i transients, whereas only low-amplitude [Ca2+]i signals have been observed in unstimulated gonadotrophs (2, 3). In parallel to spontaneous [Ca2+]i signaling, somatotrophs and lactotrophs exhibit high basal secretion, whereas basal gonadotropin secretion is low and not dependent on extracellular Ca2+ (4). Consistent with this, Ca2+ signaling and secretion in somatotrophs and lactotrophs, but not in gonadotrophs, are under dual control by positive and negative hypothalamic factors (5, 6, 7). These differences suggest that, despite the similar origin of somatotrophs, lactotrophs, and gonadotrophs, there may be differences in the ionic channels expressed in the three hormone-secreting cell types.

Many of the ionic channels in native and immortalized anterior pituitary cells have been characterized previously, including voltage-gated Ca2+ channels (VGCCs), tetrodotoxin (TTX)-sensitive and insensitive Na+ channels, voltage-gated K+ channels, Ca2+-controlled K+ channels, Cl- channels, and nonselective cationic channels (2, 3). However, it is difficult to directly compare the expression levels and the properties of the individual ionic channels from the different studies, due to differences in the species, sex, and hormonal status of the animals used, as well as the cell cultures and recording conditions. Because of this, it is still not known whether differences in the expression levels and/or ionic channel properties underlie the cell type-specific patterns of voltage-gated Ca2+ entry and hormone secretion. To address this problem, we compared the expression levels and voltage-dependent properties of the ionic channels in somatotrophs, lactotrophs, and gonadotrophs from randomly cyclic female rats under identical culture and recording conditions. A similar group of ionic channels is observed in all three cell types. However, there was a marked difference in the functional expression of the individual ionic channels between the three hormone-secreting cell types. These differences likely underlie the cell type-specific patterns of [Ca2+]i signaling and hormone secretion observed in unstimulated somatotrophs, lactotrophs, and gonadotrophs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Voltage-Gated Na+ Channels
The functional expression and voltage-dependent properties of the Na+ channels in rat somatotrophs, lactotrophs, and gonadotrophs were examined under isolated Na+ current (INa) recording conditions using the conventional whole-cell technique. In rat somatotrophs and lactotrophs, a rapidly activating and inactivating INa was observed at membrane potentials more depolarized than -37 mV and reached maximum amplitude around -7 mV (Fig. 1Go, A, B, and D). A similar INa was observed in rat gonadotrophs (Fig. 1CGo), but its peak amplitude was much greater and its current-voltage relation was shifted approximately 10 mV in the hyperpolarizing direction compared with that for somatotrophs and lactotrophs (Fig. 1DGo). In somatotrophs, lactotrophs, and gonadotrophs, application of 1 µM TTX reduced the peak INa amplitude by 99.4 ± 0.7% (n = 5), 98.2 ± 3.3% (n = 5), and 98.9 ± 1.2% (n = 5), respectively.



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Figure 1. Voltage-Gated Na+ Channels in Somatotrophs, Lactotrophs, and Gonadotrophs

Representative voltage-gated INa traces in somatotrophs (panel A, n = 5), lactotrophs (panel B, n = 5), and gonadotrophs (panel C, n = 5) elicited by 100-msec voltage steps from -87 mV to 83 mV form a holding potential of -117 mV. D, Current-voltage relation of the voltage-gated INa in all three cell types. E, Steady-state inactivation curves for the voltage-gated INa in all three cell types were generated by stepping the membrane potential to between -127 and -13 mV for 200 msec before stepping to a 100-msec command potential of -17 mV (holding potential = -97 mV). The peak INa evoked during the command potential to -17 mV in each cell type were normalized to the maximum inward current and plotted against the conditioning pulse potentials. In this and the following figures, the ionic currents were normalized to the membrane capacitance of each cell examined to compensate for the differences in the size of cells. The dashed box in this figure and in Figs. 3Go and 5Go represents the range of baseline potentials commonly observed in spontaneously active somatotrophs, lactotrophs, and gonadotrophs (54 ).

 
To determine the proportions of the TTX-sensitive INa in each cell type that are available for activation at different resting membrane potentials, the steady-state inactivation properties of the INa in all three cell types were examined using a two-pulse protocol. This protocol consisted of a series of 200-msec conditioning pulses from -127 mV to -13 mV, followed by a 100-msec test pulse to -17 mV (holding potential = -97 mV). The peak INa evoked during the test pulse was normalized to the maximal inward current and plotted against the conditioning pulse potentials, and the resulting curves were fitted with a single Boltzmann relation (Fig. 1EGo). In somatotrophs, the membrane potential at which there is 50% of the maximal current (E1/2) available for activation is -72 mV. In lactotrophs and gonadotrophs, the E1/2 values were similar and were approximately 10 mV more depolarized than that observed in somatotrophs.

Voltage-Gated Ca2+ Channels
The functional expression and voltage-dependent properties of the Ca2+ channels in rat somatotrophs, lactotrophs, and gonadotrophs were examined under isolated Ca2+ current (ICa) recording conditions using the conventional whole-cell technique. The ICa in each cell type was examined by applying a series of 400-msec depolarizing voltage steps from -97 mV to +83 mV in 10-mV increments (holding potential = -97 mV). In all three cell types, a transient ICa was observed in response to the depolarizing voltage steps. In somatotrophs, the transient ICa was activated by voltage steps more depolarized than -77 mV and reached maximum amplitude between -27 and -17 mV (Fig. 2Go, A and B). In the other two cell types, the activation threshold of the transient current was shifted approximately 10 mV in the depolarizing direction (Fig. 2Go, A and B). In addition, the peak of the transient ICa-voltage relationship was shifted more than 20 mV in the depolarizing direction compared with that in somatotrophs (Fig. 2BGo). To determine the density of the transient ICa in each cell type, the peak ICa evoked by voltage steps to -47 mV (holding potential = -97 mV) was analyzed. This membrane potential was used because it activates a transient ICa in all three cell types with only minimal activation of a sustained ICa (Fig. 2AGo). The mean peak transient ICa densities at -47 mV in somatotrophs, lactotrophs, and gonadotrophs were -6.6 ± 0.8 picoamperes/picofarads (pA/pF) (n = 20), -2.2 ± 0.3 pA/pF (n = 16), and -2.7 ± 0.6 pA/pF (n = 14), respectively.



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Figure 2. Voltage-Gated Ca2+ Channels in Somatotrophs, Lactotrophs, and Gonadotrophs

A, Representative voltage-gated ICa traces in somatotrophs (n = 20), lactotrophs (n = 16), and gonadotrophs (n = 14) elicited by 400-msec voltage steps from a holding potential of -97 mV to -77, -47, -27, and -7 mV are shown. B, Current-voltage relation of the peak (open circles, 0–25 msec) and sustained (filled circles, 390–400 msec) voltage-gated ICa in all three cell types.

 
The transient ICa in somatotrophs, lactotrophs, and gonadotrophs was followed by slow-inactivating ICa. In all three cell types, the sustained ICa was activated by voltage steps more depolarized than -57 mV and reached a maximum amplitude around +3 mV (Fig. 2Go, A and B). In addition to the similar current-voltage relationship of the sustained ICa, dihydropyridine agonists and antagonists had a similar effect on the sustained ICa amplitude in all three cell types (Table 1Go). To compare the density of the slow-inactivating ICa in each cell type, the sustained ICa (390–400 msec) evoked by voltage-steps to +3 mV was analyzed. In somatotrophs, lactotrophs, and gonadotrophs the mean sustained ICa densities were -6.8 ± 0.8 pA/pF (n = 20), -4.5 ± 0.9 pA/pF (n = 16), and -9.2 ± 1.7 pA/pF (n = 14), respectively.


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Table 1. Effects of L-Type Ca2+ Channel Agonists and Antagonists on Sustained ICa in Somatotrophs, Lactotrophs, and Gonadotrophs

 
To determine the proportions of the total voltage-gated ICa in each cell type that is available for activation at various different resting membrane potentials, the steady-state inactivation properties of the ICa were examined using a two-pulse protocol. This protocol consisted of a conditioning pulse ranging from -127 mV to -13 mV for 400 msec, after which a 200-msec test pulse to -7 mV was applied (Fig. 3Go, A–C). The normalized test current in each cell type was plotted against the conditioning pulse potentials, and the resulting curve was fitted with a single Boltzmann relation (Fig. 3DGo). In somatotrophs, E1/2 was -56 mV (slope factor = 6.5). The E1/2 in lactotrophs and gonadotrophs were -34 mV (slope factor = 7) and -40 mV (slope factor = 4), respectively, which were more than 15 mV more depolarized than that in somatotrophs. The more pronounced steady-state inactivation of the total ICa in somatotrophs compared with that in lactotrophs and gonadotrophs is consistent with the greater expression of the transient ICa in somatotrophs.



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Figure 3. Steady-State Inactivation of the total ICa in Somatotrophs, Lactotrophs, and Gonadotrophs

A–C, Steady-state inactivation curves for the voltage-gated ICa in each cell type were generated by stepping the membrane potential to between -127 and -3 mV for 400 msec before stepping to a 200-msec command potential of -7 mV (holding potential = -97 mV). Representative traces of the remaining current elicited during the command potential after conditioning pulses between -77 mV and -17 mV are shown. D, The peak ICa evoked during the command potential to -7 mV in all three cell types were normalized to the maximum inward current and plotted against the conditioning pulse potentials. The data for each cell type were fitted with a single Boltzmann relation.

 
Voltage-Gated K+ Channels
The functional expression and voltage-dependent properties of the voltage-gated K+ channels in rat somatotrophs, lactotrophs, and gonadotrophs were examined under isolated K+ current (IK) recording conditions using the perforated-patch technique. To exclude Ca2+-sensitive IK (IK(Ca)), extracellular Ca2+ entry through VGCCs was blocked by addition of 200 µM Cd2+ to the bath solution. The total voltage-gated IK in each cell type was examined by the application of a 500-msec holding potential to -130 mV before giving a series of 1.5-sec depolarizing voltage steps from -90 mV to +90 mV (Fig. 4AGo; upper panels). In rat somatotrophs, both the peak (0–25 msec) and sustained (1.4–1.5 sec) IK were activated at membrane potentials more depolarized than -30 mV. Once activated, the total voltage-gated IK inactivated slowly during the 1.5-sec depolarizing voltage steps (Fig. 4Go, A and D). In lactotrophs and gonadotrophs, the peak IK activated at membrane potentials more depolarized than -50 mV, whereas the sustained IK activated at membrane potentials more depolarized than -30 mV. Unlike somatotrophs, the total voltage-gated IK in lactotrophs and gonadotrophs was characterized by a fast- and slow-inactivating IK (Fig. 4Go, A and D).



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Figure 4. Voltage-Gated K+ Channels in Somatotrophs, Lactotrophs, and Gonadotrophs

A, Representative voltage-gated IK traces in somatotrophs (n = 5), lactotrophs (n = 5), and gonadotrophs (n = 8) elicited by 1.5-sec voltage steps from -90 to 90 mV in 20-mV increments from a holding potential of -130 mV. B, Representative voltage-gated IK traces in somatotrophs (n = 5), lactotrophs (n = 5), and gonadotrophs (n = 8) elicited by 1.5-sec voltage steps from -90 to 90 mV in 20-mV increments from a holding potential of -40 mV. C, A transient IK in all three cell types was isolated by a point-by-point subtraction of the current traces in panels A and B. D, Current-voltage relationship of the peak (0–25 msec; left panel), sustained (1.49–1.50 sec; middle panel) and subtracted (A - B; right panel) IK in all three cell types. To block IK(Ca), 200 µM CdCl2 was added to the bath solution to block voltage-gated Ca2+ entry.

 
To isolate the slow-inactivating IK, the transient IK was eliminated by the application of a 500-msec holding potential to -40 mV before the 1.5-sec depolarizing voltage steps from -90 mV to +90 mV (Fig. 4BGo). Under these conditions, a slow-inactivating IK was observed in somatotrophs and lactotrophs (Fig. 4BGo). A similar slow-inactivating IK was observed in gonadotrophs in response to voltage steps between -40 and 20 mV. However, a small fast-inactivating IK was maintained at more depolarized membrane potentials, which may be due to incomplete inactivation of the transient IK in gonadotrophs by the conditioning pulse. To compare the densities of the slow-inactivating IK in each cell type, the sustained IK evoked by voltage steps to +90 mV (holding potential of -90 mV) was analyzed. The sustained IK density in lactotrophs was 147 ± 15 pA/pF (n = 5), which was significantly (P < 0.05) smaller than that in somatotrophs (254 ± 32 pA/pF; n = 5) and gonadotrophs (282 ± 32 pA/pF; n = 8).

We next compared the transient IK in each cell type by subtracting the IK elicited during the various voltage steps after a holding potential to -40 mV from the total IK (Fig. 4CGo). In rat somatotrophs, a small transient IK was evoked by membrane potential steps more depolarized than -30 mV, reaching a peak amplitude of 67 ± 18 pA/pF at +90 mV (n = 5). In contrast, a large transient IK was observed in lactotrophs (n = 5) and gonadotrophs (n = 8; Fig. 4Go, C and D; right panel). The transient IK in these cell types activated at membrane potentials more depolarized than -50 mV and reached maximum amplitude at +90 mV of 343 ± 73 pA/pF and 389 ± 82 pA/pF at +90 mV, respectively.

To determine the proportions of the total voltage-gated IK in each cell type that is available for activation at various different membrane potentials, the steady-state inactivation properties of the IK were examined using a two-pulse protocol. This protocol consisted of a series of 1.5-sec conditioning pulses from -130 mV to -10 mV, followed by a 400-msec test pulse to +90 mV (Fig. 5AGo). The representative IK tracings evoked by the two-pulse protocol are shown in Fig. 5Go, A–C. The normalized test current in each cell type was plotted against the conditioning pulse potentials (Fig. 5DGo). A single Boltzmann relation could not be fitted to the data, further indicating the presence of a fast- and slow-inactivating component in each of the three cell types. However, the magnitude of steady-state IK inactivation in somatotrophs was much less than that observed in the other two cell types, which is consistent with the low expression levels of the transient IK in somatotrophs.



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Figure 5. Steady-State Inactivation of the Total IK in Somatotrophs, Lactotrophs, and Gonadotrophs

A–C, Steady-state inactivation curves for the peak voltage-gated IK in each cell type were generated by stepping the membrane potential to between -130 and -10 mV for 1.5 sec before stepping to a command potential of 90 mV (holding potential = -90 mV). Representative traces of the remaining current elicited during the command potential after conditioning pulses to -130, -110, -90, -70, -50, -30, -10, and 10 mV are shown. D, The peak IK evoked during the command potential to +90 mV all three cell types were normalized to the maximum inward current and plotted against the conditioning pulse potentials.

 
Ca2+-Activated K+ Channels
Voltage-gated Ca2+ entry was used to examine the expression of IK(Ca) in somatotrophs, lactotrophs, and gonadotrophs. To activate VGCCs, a modified two-step protocol was used. This protocol consisted of an initial 100-msec voltage-step to -10 mV (holding potential = -90 mV) during which VGCCs were activated (Fig. 6AGo; left panel). This Ca2+-influx step was immediately followed by a 500-msec test pulse to +80 mV, during which the evoked IK was monitored. As +80 mV is near the reversal potential for Ca2+ under our experimental conditions, there should be minimal Ca2+ entry during this step. Consistent with this, in the absence of the Ca2+-influx step, there was little to no change in [Ca2+]i, whereas in the presence of a Ca2+-influx step alone or in combination with the test pulse there was a marked increase in [Ca2+]i in all three cell types (Fig. 7Go). In addition, the IK evoked by the test pulse to +80 mV in the absence of the Ca2+-influx step was similar in Ca2+-containing and Ca2+-deficient medium (Table 2Go).



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Figure 6. Identification of IK-Ca in Somatotrophs, Lactotrophs, and Gonadotrophs

A–C, Left panels, Representative IK traces in somatotrophs (n = 13), lactotrophs (n = 15), and gonadotrophs (n = 9) evoked by the two-step protocol (A, upper panel) in Ca2+-containing (control) and Ca2+-deficient (-Ca2+) medium. The two-step protocol consisted of a 100-msec condition pulse to -10 mV, to activated voltage-gated Ca2+ entry, followed by a 500-msec test pulse to +90 mV, during which the peak IK was monitored. A–C, Right panels, The net IK-Ca activated by the two-step protocol in each cell type was isolated by subtracting the current evoked in the presence of Ca2+-deficient medium from that in Ca2+-containing medium. The mean ± SEM values of the peak IKCa evoked during the test pulse are shown.

 


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Figure 7. Profile of [Ca2+]i in Cells Exposed to One- and Two-Step Protocol.

A–D, Representative [Ca2+]i traces without (solid line) and with (dotted line) the application of a 100-msec Ca2+-influx step to -10 mV before the application of a 500-msec step to a test potential (TP) of +80 mV or by the Ca2+-influx step (CIS) alone (thick line) from a holding potential = -90 mV in somatotrophs (n = 17), lactotrophs (n = 12), and gonadotrophs (n = 12). The net change in [Ca2+]i (mean ± SEM) evoked by the TP, CIS, and TP + CIS are shown in the right panel. The perforated patch-clamp recording configuration was used to control the membrane potential in cells preloaded with 0.5 µM Indo 1-AM.

 

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Table 2. Peak IK in Somatotrophs, Lactotrophs, and Gonadotrophs Evoked by a 500-msec Voltage Step from -90 mV to +80 mV in Ca2+-Containing and Ca2+-Deficient Medium

 
In the presence of a 100-ms Ca2+-influx step, the peak IK evoked during the test pulse was reduced by extracellular Ca2+ removal in somatotrophs, lactotrophs, and gonadotrophs (Fig. 6Go, A–C, and Table 3Go), indicating IK(Ca) values are expressed in all three cell types. To compare IK(Ca) activation between the three cell types, the IK evoked in Ca2+-deficient medium was subtracted from that in Ca2+-containing medium (Fig. 6Go, A–C, right panels). These results clearly indicate that IK(Ca) activation by voltage-gated Ca2+ entry was greatest in somatotrophs and smallest in gonadotrophs.


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Table 3. Effects of Ca2+-Deficient Medium and Ca2+-Sensitive K+ Channel Blockers and Activators on the Peak IK in Somatotrophs, Lactotrophs, and Gonadotrophs

 
The differences in IK(Ca) activation between the three cell types may be due to differences in the ability of the Ca2+ influx step to drive voltage-gated Ca2+ entry and increase [Ca2+]i. To test this, we compared the increase in [Ca2+]i evoked by the two-step protocol in each cell type (Fig. 7Go). In somatotrophs and gonadotrophs, the two-step protocol increased the [Ca2+]i by 160 ± 18 nM (n = 17) and 164 ± 19 nM (n = 12), respectively. In lactotrophs, a smaller increase in [Ca2+]i of 122 ± 13 nM (n = 12) was evoked by the two-step protocol. These results are consistent with the smaller amplitude noninactivating ICa in lactotrophs compared with the other two cell types (Fig. 2Go). Nevertheless, these results indicate that the small amplitude IK(Ca) observed in gonadotrophs is not due to the inability of voltage-gated Ca2+ entry to increase [Ca2+]i. Therefore, the differences between IK(Ca) activation in the three cell types appears to be due to differences in channel expression and not the capacity of the Ca2+-influx step to drive changes in [Ca2+]i.

The dependence of IK(Ca) activation on voltage-gated Ca2+ influx was further confirmed in experiments with dihydropyridine agonist and antagonists. As shown in Fig. 8Go, A and C, addition of the L type Ca2+ channel blocker, nifedipine, significantly decreased the amplitude of the IK in all three cell types studied. Furthermore, Bay K 8644, an L-type Ca2+ channel agonist, significantly increased the amplitude of IK in all three cell types (Fig. 8Go, A and C). Consistent with the differential expression of Ca2+-activated K+ channels in the three cell types, the effects of nifedipine and Bay K 8644 were small in gonadotrophs, and larger in lactotrophs and somatotrophs.



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Figure 8. Dihydropyridine Sensitivity of ICa and IK-Ca in Pituitary Cells

Under isolated IK recording conditions, the effects of 1 µM nifedipine (A) and 1 µM (–)-Bay K 8644 (B) on the IK amplitude evoked during the TP by 50-msec (somatotrophs) or 100-msec (lactotrophs and gonadotrophs) CIS were examined, and the representative current recordings are shown. C, The peak IK amplitude during the test pulse in the presence of 1 µM nifedipine or 1 µM (-)-Bay K 8644 were normalized to the levels reached in the presence of the vehicle alone (control) and expressed as the mean ± SEM for somatotrophs (n = 3), lactotrophs (n = 5), and gonadotrophs (n = 3). Asterisks indicate significant differences (P < 0.05) compared with control values.

 
To determine the IK(Ca) subtype activated by voltage-gated Ca2+ entry in somatotrophs, lactotrophs, and gonadotrophs, we used selective blockers and stimulators of BK and SK channels. The specific BK channel blockers, charybdotoxin (CTX; 100 nM), iberiotoxin (IBTX; 100 nM), and paxilline (1 µM), markedly reduced the IK in all three cell types (Fig. 9Go, A–C and Table 3Go). In addition, the BK channel activator, NS 1619 (30 µM), increased IK amplitude in somatotrophs and lactotrophs, but not in gonadotrophs (Fig. 9DGo and Table 3Go). Unlike the BK channel blockers, the SK channel blocker, apamin, had no effect on the IK evoked by the 100-msec Ca2+-influx step in the majority of somatotrophs (n = 27) and lactotrophs (n = 11) examined. In the remaining somatotrophs and lactotrophs, apamin significantly reduced the IK (Fig. 9EGo and Table 3Go). In all gonadotrophs examined (n = 10), apamin had no effect on the IK evoked by the 100-msec Ca2+-influx step (Fig. 9EGo and Table 3Go). This was not due to the absence of SK channels in these cells, as GnRH simulated ISK in all gonadotrophs (data not shown).



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Figure 9. Pharmacological Identification of BK Channels in Pituitary Cells

A–D, Representative current traces showing the effects of 100 nM IBTX (A), 100 nM CTX (B), 1 µM paxilline (C), 30 µM NS 1619 (D), and 100 mM apamin (E), on the IK in somatotrophs, lactotrophs, and gonadotrophs. Averaged data for each cell type are shown in Table 3Go.

 
In the presence of apamin and CTX, IBTX, or paxilline, no further reduction in IK was observed when the cells were perifused with Ca2+-deficient medium (data not shown). These results indicate that IK(Ca) activation by voltage-gated Ca2+ influx in somatotrophs, lactotrophs, and gonadotrophs is predominantly mediated by BK channels. It should be noted, however, that the inability of apamin to significantly reduce the IK in a majority of the cells examined is most likely due to the relatively small current generated by its activation and not the lack of expression in each cell type. In addition, other Ca2+-sensitive currents that are expressed in pituitary cells, such as Ca2+-activated Cl- channels (8, 9), may be masked by the much larger IK in these cells.

To determine the duration of voltage-gated Ca2+ influx required to activate IBK in each cell type, we varied the duration of the Ca2+-influx step and measured the peak IK amplitude during the subsequent 500-msec voltage step to +80 mV. To isolate BK channels from SK channels, 100 nM apamin was added to the extracellular medium. In somatotrophs, 5-msec Ca2+-influx steps were sufficient to activate IBK, whereas 10-msec steps were required in lactotrophs. In both cell types, the peak IK increased progressively in response to incremental increases in the duration of the Ca2+-influx step (Fig. 10Go, A–C). This increase was not observed in Ca2+-deficient medium (Fig. 10Go, B and C) or in cells preloaded with BAPTA-AM (Fig. 10CGo). In gonadotrophs, Ca2+-influx steps greater than 75 msec were required to activate IBK (Fig. 10Go, A–C). In addition, no increase in IBK activation was observed in response to further increases in the duration of voltage-gated Ca2+ influx (Fig. 10CGo). These results suggest that brief periods of voltage-gated Ca2+ influx are sufficient to activate BK channels in somatotrophs and lactotrophs, whereas prolonged Ca2+ influx is required in gonadotrophs. Moreover, it appears that increasing the duration of voltage-gated Ca2+ influx recruits more BK channels in somatotrophs and lactotrophs, but not gonadotrophs.



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Figure 10. Dependence of IBK Activation on Duration of Voltage-Gated Ca2+ Influx

The effects of increasing the duration of VGCC entry on IK activation were examined by incremental increases in the duration of Ca2+ influx step from 0 to 100 msec, followed by a test pulse to +80 mV for 500 msec. Representative current tracings recorded in Ca2+-containing (A) or Ca2+-deficient medium (B) are shown for somatotrophs (n = 13), lactotrophs (n = 12), and gonadotrophs (n = 12). C, Peak IK amplitude (mean ± SEM) during the test pulse after 0- to 150-msec conditioning pulses in Ca2+-containing medium (filled circles), Ca2+-deficient medium (open circles), and in cells preloaded with BAPTA-AM (filled triangles). Asterisks indicate a significant difference compared with the peak IK evoked in the absence of a depolarizing pulse. All experiments were performed in the presence of 100 nM apamin to block SK channel activation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Despite the similar origin of somatotrophs, lactotrophs, and gonadotrophs, these cells differ with respect to their pattern of basal hormone secretion. Specifically, basal gonadotropin is low compared with basal GH and PRL secretion. Earlier studies have indicated that these differences in basal hormone secretion are due to differences in spontaneous Ca2+ signaling among the three cell types. Unlike basal GH and PRL secretion, basal gonadotropin secretion is not dependent on the presence of extracellular Ca2+ or modified by increases in the extracellular Ca2+ concentration (4). In addition, application of voltage-gated Ca2+ channel blockers reduces basal GH and PRL secretion, but not basal LH or FSH secretion (4, 25). Consistent with these differences in the apparent role of voltage-gated Ca2+ channels in controlling basal hormone secretion, the profile of spontaneous Ca2+ signaling among the three cell types is also different. In rat lactotrophs and somatotrophs, spontaneous and high amplitude Ca2+ signals have been observed, whereas only low amplitude Ca2+ signals have been observed in spontaneously active rat gonadotrophs (2, 3, 4, 25, 35). These differences in the extracellular Ca2+ dependency and voltage-gated Ca2+ channel involvement in determining the pattern of basal hormone secretion and the cell type-specific patterns of spontaneous Ca2+ signaling suggest that there are differences in the level and/or composition of ionic channel expression and the underlying electrical activity between rat gonadotrophs, lactotrophs, and somatotrophs.

In this study, we compared the expression levels of different ionic channels between pituitary somatotrophs, lactotrophs, and gonadotrophs. As a measure of the functional expression levels of the ionic channels in each cell type, the isolated currents were analyzed. The results indicate a quantitative rather than qualitative difference in the expression pattern for the major ionic currents in pituitary cells. All three cell types express transient and sustained voltage-gated Ca2+ channels, TTX-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+-sensitive K+ channel subtypes. Lactotrophs and somatotrophs exhibited low expression levels of TTX-sensitive Na+ channels and high expression levels of BK channel compared with those observed in gonadotrophs. The expression of the transient voltage-gated K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient VGCCs was higher in somatotrophs than in lactotrophs and gonadotrophs.

TTX-sensitive Na+ channels have been previously identified in gonadotrophs (10, 11), lactotrophs (12), and somatotrophs (13), as well as other pituitary cell types (14, 15, 16), indicating that these channels are common among mammalian pituitary cells. Our results extended these findings by demonstrating that the level of Na+ channel expression is much greater in rat gonadotrophs than the other two cell types. However, it is unlikely that the difference in the expression of these channels is relevant for the control of spontaneous electrical activity (12, 17), Ca2+ signaling (17), and basal hormone secretion (16, 18) in these cells. The lack of TTX-sensitive Na+ channel involvement in controlling membrane excitability and secretion is most likely due to the inactivation of a large proportion of these channels at the resting membrane potential in these cells (Fig. 1Go and Ref. 10). Consistent with this, GnRH-induced transient membrane hyperpolarization in rat gonadotrophs is required to remove the steady-state inactivation of TTX-sensitive Na+ channels before they can contribute to action potential (AP) firing (10). This TTX sensitivity during agonist-induced AP firing in gonadotrophs is important for sustained Ca2+ signaling through VGCCs and the refilling of endoplasmic reticulum Ca2+ stores. There are, however, two exceptions. First, in a fraction of ovine gonadotrophs, these channels are responsible for AP generation (19). Second, two lactotroph subpopulations have been identified that differ with respect to their level of Na+ channel expression, and only in lactotrophs expressing high levels of Na+ channels did TTX application abolish basal hormone secretion (20). In the present study, we did not observe a significant difference in the level of Na+ channel expression in the lactotrophs identified by our cell separation and identification protocol.

The expression of both inactivating and noninactivating VGCCs in rat gonadotrophs (10, 21, 22), somatotrophs (13), and lactotrophs (13), as well as immortalized pituitary cells (14, 15, 23, 24), has been well documented. The inactivating ICa is mediated by the low voltage-activated or transient (T)-type Ca2+ channel. Although we observed T-type ICa in all three cell types examined, it was more prominent in somatotrophs than in lactotrophs and gonadotrophs. A similar conclusion was reached in a study that directly compared VGCC expression between somatotrophs and lactotrophs. The prominent expression of T-type Ca2+ channels in somatotrophs is reflected by their contribution to the generation of the high amplitude [Ca2+]i transients in spontaneously active somatotrophs (25), whereas its role in other native anterior pituitary cells is not known. The noninactivating ICa in pituitary cells is mediated by dihydropyridine-sensitive (L-type) and -insensitive, high-voltage activated Ca2+ channels (10, 21, 24, 26). Unlike the T-type Ca2+ channel, the current voltage-relationship of the sustained ICa in all three cell types was similar. However, the sustained ICa density was higher in somatotrophs and gonadotrophs than in lactotrophs. Despite the different densities of the sustained ICa, it is essential to the generation of both spontaneous and agonist-induced AP-driven Ca2+ entry in all native and immortalized pituitary cells (4, 27, 28). In addition, the L-type VGCC has been demonstrated to contribute to the regulation of basal and agonist-stimulated GH and PRL secretion, as well as agonist-induced gonadotropin secretion (28, 29).

In general, the VGCC-dependent rise in [Ca2+]i is sufficient to trigger activation of several Ca2+-sensitive channels. One such channel is the BK-type K+ channel, the expression of which has been previously demonstrated in immortalized anterior pituitary cells (30, 31, 32), and native intermediate pituitary cells (33). Our studies indicate that BK channels are also expressed in native anterior pituitary cells, and that they are coupled to voltage-gated Ca2+-influx in all three cell types examined. Moreover, by comparing BK channel activation between somatotrophs, lactotrophs, and gonadotrophs under identical culture and recording conditions, it was demonstrated that BK channel activation was much greater in somatotrophs than in gonadotrophs. Due to the similarities in the voltage-gated ICa density and change in [Ca2+]i evoked by the Ca2+-influx step between the three cell types, the differences in IBK activation are most likely due to differences in BK channel expression levels.

In other excitable cells, the colocalization of BK channels with VGCCs facilitates spike repolarization, which limits AP-driven Ca2+ influx. BK channel activation can also influence the frequency of AP-driven [Ca2+]i transients by slowing the pacemaker depolarization (34). In native anterior pituitary cells, the role of BK channels in shaping the frequency and duration of AP-driven Ca2+ entry is not known. Based on our results, we would expect that the relatively high levels of BK channel expression in somatotrophs and lactotrophs would limit AP-driven Ca2+ influx compared with that in gonadotrophs, which exhibit the lowest levels of BK channel expression. However, previous studies have demonstrated that the duration of the AP waveform is longer in somatotrophs and lactotrophs (100–500 msec) than in gonadotrophs (10–100 msec) (10, 35, 36, 37). In addition, both the amplitude and duration of the spontaneous, extracellular Ca2+-dependent [Ca2+]i transients are greater in somatotrophs and lactotrophs than in gonadotrophs (2). It is unlikely that the prolonged duration of AP-driven Ca2+ entry in somatotrophs and lactotrophs is due to the inability of AP firing to activate BK channels, as short Ca2+ influx steps (<25 msec) were sufficient to activate IBK in both cell types (Fig. 10Go). Thus, whether BK channels have an atypical role in regulating the pattern of AP firing and Ca2+ signaling in anterior pituitary cells requires further studies.

All three pituitary cell types also express SK channels (31, 38, 39, 40). However, in gonadotrophs SK channels did not appear to be coupled to voltage-gated Ca2+ influx under the conditions used in our study. Similarly, voltage-gated Ca2+ influx activated SK channels in only a small fraction of the somatotrophs and lactotrophs examined. This may be due to the lack of SK channel expression in some somatotroph and lactotroph subpopulations. Consistent with this, activation of Ca2+-mobilizing TRH receptors leads to activation of SK channels and the concomitant membrane hyperpolarization in GH3 cells (32), but only a small fraction of lactotrophs exhibited a similar response (41). It is also possible that the SK channels in pituitary cells, as in other cell types (reviewed in Ref. 42), are colocalized with intracellular Ca2+ release sites and can be activated only by Ca2+-mobilizing receptors, sustained voltage-gated Ca2+ entry, and/or Ca2+-induced Ca2+ release. For example, in GnRH-secreting neurons, agonist-induced Ca2+ mobilization and the concomitant increase in firing frequency are needed to activate SK channels (43). Similarly, in GH3 cells, SK channel activation requires high-frequency firing, prolongation of APs by voltage-dependent K+ channel inhibitors, or release of Ca2+ from intracellular Ca2+ stores (31). The lack of a detectable apamin-sensitive IK in a majority of the three cell types examined may also be due to its relatively small size compared with the voltage-gated IK and the IBK in these cells. Other Ca2+-sensitive channels, such as Cl- channels [known to be expressed in AtT-20 immortalized cells (8) and native lactotrophs (9)] may also be masked.

Several different voltage-gated K+ channel subtypes have been identified and characterized in somatotrophs, lactotrophs, and gonadotrophs (44, 45), as well as immortalized pituitary cells (14, 15). One such channel is the transient, 4-AP-sensitive (A-type) K+ channel. Direct comparison of the three anterior pituitary cells examined in this study indicates that the expression level of the A-type K+ channel is much higher in lactotrophs and gonadotrophs than in somatotrophs. In contrast, these channels have been observed in ovine somatotrophs and may contribute to AP firing and hormone secretion (46). The participation of the A-type K+ channel in regulating AP firing in other anterior pituitary cell types is also unclear. In rat lactotrophs, for example, they do not appear to participate in AP generation (37), which may be due to their prominent inactivation at the resting membrane potential in these cells. The participation of the delayed rectifying K+ channel during AP firing in anterior pituitary cells is also not clear. In immortalized cells, inhibition of this channel by tetraethylammonium (TEA) increased the duration of the AP (37) and the amplitude of the spontaneous [Ca2+]i transients (47), whereas in native rat lactotrophs, TEA did not alter the pattern of AP firing (37). Further studies are required to elucidate the role of both the A-type and delayed rectifying K+ channels in the regulation of AP-driven Ca2+ signaling in the different anterior pituitary cell types.

Our study was focused on the ionic channels involved in the formation of the AP waveform. Other channels have also been identified in pituitary cells, and they contribute to pacemaking activity in these cells. For example, a TTX-insensitive INa has been observed in somatotrophs (48) and lactotrophs (12) and is critical for maintaining the membrane potential near the threshold for AP firing. In addition, inward rectifier K+ channels, including the erg-like K+ channel, have been identified in both native (36, 49) and immortalized pituitary cells (50). They contribute to the regulation of both spontaneous and agonist-modulated AP-driven Ca2+ entry. The M-type K+ channel has also been identified in pituitary cells (41), as well as several ATP-gated P2X receptor channels (51). Preliminary data also suggested the presence of cyclic nucleotide-gated channels in pituitary cells, which may contribute to pacemaking activity (25).

In summary, we examined the cell-type specific expression of the major ionic currents in somatotrophs, lactotrophs, and gonadotrophs that contribute to AP-driven Ca2+ entry. All experiments were done under identical experimental conditions on cells from randomly cycling adult female rats. This allowed us to directly compare the expression levels and voltage-dependent properties of several ionic channels between the three anterior pituitary cell types. Our results demonstrate that, although these cells originate from the same precursor cell, they exhibit cell type-specific patterns of ionic channel expression. In particular, the marked differences in BK channel and transient (A-type) K+ channel expression between the three cell types are excellent candidates for future investigations into the cell type-specific patterns of spontaneous and receptor-controlled membrane excitability, Ca2+ signaling, and hormone secretion.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pituitary Cell Culture and Cell Identification
Anterior pituitary glands were excised from female Sprague Dawley rats (Taconic Farms, Inc., Germantown, NY) and dispersed into single cells using a trypsin/DNase (Sigma, St. Louis, MO) cell dispersion procedure as described previously (51). Enriched somatotroph (>90%, estimated by immunocytochemistry using specific antibodies provided by the National Hormone and Pituitary Program and Dr. Parlow) and lactotroph (>65%) populations were obtained using a discontinuous Percoll density-gradient cell-separation procedure as described previously (51). Pituitary somatotrophs and lactotrophs were further identified using a combination of cell separation techniques, their distinct morphology, and responses to GHRH and TRH, respectively. Gonadotrophs were initially identified by their distinct morphology and subsequent to experimentation by their unique oscillatory IK and/or [Ca2+]i response to GnRH, the specific agonist for these cells.

Electrophysiological Recordings
Voltage-gated INa and ICa were measured using regular whole-cell recording techniques, whereas voltage-gated IK and IK(Ca) were measured using perforated-patch recording techniques (52). All voltage-clamp recordings were performed at room temperature using an Axopatch 200 B patch-clamp amplifier (Axon Instruments, Foster City, CA) and were low-pass filtered at 2 kHz. A series resistance of < 20 M{Omega} was reached 10 min after the formation of a gigaohm seal (seal resistance > 5 G{Omega}). When necessary, series resistance compensation was optimized and all current recordings were corrected for linear leakage and capacitance using a P/-N procedure. An average membrane capacitance (Cm) of 4.6 ± 0.2 pF (n = 48), 5.9 ± 0.2 pF (n = 47), and 7.5 ± 0.2 pF (n = 68) was recorded in somatotrophs, lactotrophs, and gonadotrophs, respectively. Pulse generation and data acquisition were done with a PC equipped with a Digidata 1200 A/D interface in conjunction with Clampex 8 (Axon Instruments).

Simultaneous Measurement of [Ca2+]i and IK
To simultaneously monitor [Ca2+]i and IK the cells were incubated for 15 min at 37 C in phenol red-free medium 199 containing Hanks’ salts, 20 mM sodium bicarbonate, 20 mM HEPES, and 0.5 µM indo-1 AM (Molecular Probes, Inc., Eugene, OR). The coverslips with cells were then washed twice with modified Krebs-Ringer’s solution containing (in mM): 120 NaCl, 4.7 KCl, 2.0 CaCl2, 2 MgCl2, 0.7 MgSO4, 10 HEPES, 10 glucose (pH adjusted to 7.4 with NaOH) and mounted on the stage of an inverted epifluorescence microscope (Nikon, Melville, NY). A photon counter system (Nikon) was used to simultaneously measure the intensity of light emitted at 405 nm and at 480 nm after excitation at 340 nm. Background intensity at each emission wavelength was corrected. Perforated-patch recording techniques (see above) were used to control the membrane potential and monitor IK in the voltage-clamp recording mode. The data were digitized at 4 kHz using a PC equipped with the Clampex 8 software package in conjunction with a Digidata 1200 A/D converter (Axon Instruments). The [Ca2+]i was calibrated in vivo as described by Kao (53). Briefly, Rmin was determined by exposing the cells to 10 µM Br-A23187 in the presence of Krebs-Ringer’s solution with 2 mM EGTA and 0 mM Ca2+ for 60 min; 15 mM Ca2+ was then added to determine Rmax. The values used for Rmin, Rmax, Sf,480/Sb,480, and dissociation constant (Kd) were 0.677, 2.9, 2.473, and 230 nM, respectively.

Chemicals and Solutions
For all IK recordings, the extracellular medium contained modified Krebs-Ringer salts containing (in mM): 120 NaCl, 4.7 KCl, 2.0 CaCl2, 2 MgCl2, 0.7 MgSO4, 10 HEPES, 10 glucose and 1 µM TTX (pH adjusted to 7.4 with NaOH) and the pipette solution contained (in mM): 50 KCl, 90 K-aspartate, 1 MgCl2, and 10 HEPES (pH adjusted to 7.2 with KOH). To isolate the voltage-gated IK, the extracellular medium contained 200 µM Cd2+ and 100 µM TTX. In some experiments, extracellular Ca2+ was replaced with equimolar Mg2+. To isolate INa and ICa, conventional whole-cell recording techniques were used as previously described (52). The extracellular medium contained Krebs-Ringer’s solution with 20 mM TEA and 2 mM CaCl2 (pH adjusted to 7.4 with NaOH) and the pipette contained (in mM); 120 CsCl, 20 TEA-Cl, 4 MgCl2, 10 EGTA, 9 glucose, 20 HEPES, 0.3 Tris-GTP, 4 Mg-ATP, 14 CrPO4, and 50 U/ml creatine phosphokinase (pH adjusted to 7.2 with Tris base). To isolate the INa or ICa, 200 µM Cd2+ or 1 µM TTX was added to extracellular medium, respectively. All reported membrane potentials were corrected on line for a liquid junction potential of 10 mV between the pipette and bath solution, except for INa and ICa, which required a correction of 7 mV. The bath contained <500 µl of saline and was continuously perfused at a rate of 2 ml/min using a gravity-driven superfusion system. Stock solutions of TTX, isobutylmethylxanthine, and CTX were prepared in double-distilled, deionized water, whereas stock solutions of paxilline, nifedipine and S(–)-Bay K 8644 were prepared in dimethylsulfoxide. All chemicals were obtained from Sigma-Aldrich Corp. (St. Louis, MO). In some experiments, the cells were loaded with 1 µM BAPTA-AM (Sigma-Aldrich Corp.) at 37 C for 45 min.

Data Analysis
Data analysis was performed using Clampfit (Axon Instruments). In some cases, the current-voltage relations were fit with a single Boltzmann relation: I/Imax = Imax + exp[(E - E1/2)/k]; where Imax is the maximum current, E is the command potential, E1/2 is the membrane potential at which there is 50% of the maximal current, and k is the slope factor. The results shown are typical tracing or means ± SEM for at least five recordings. Differences between groups were considered to be significant with P < 0.05 or higher, calculated by paired t test or ANOVA, followed by Fisher’s least significant differences test.


    FOOTNOTES
 
Address requests for reprints to: Dr. Fredrick Van Goor, c/o Stanko S. Stojilkovic, Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 6A36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov

Received for publication July 10, 2000. Revision received March 27, 2001. Accepted for publication April 2, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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