Introduction

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In cells operated by calcium-mobilizing agonists, intracellular Ca²⁺ release is coupled to extracellular Ca²⁺ influx. The coordinate action of these two pathways provides a sustained rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) during prolonged agonist stimulation (Putney and Bird, 1993), which is critical to normal cell function. In nonexcitable cells, capacitative calcium entry accounts for agonist-induced Ca²⁺ influx (Putney and Bird, 1993). This pathway is also operative in some excitable cell types and is functionally integrated with voltage-gated Ca²⁺ influx (VGCI) (Berridge, 1998). Coupling of Ca²⁺ mobilization and influx pathways has been extensively studied in pituitary lactotrophs and GH-immortalized lactosomatotrophs (Stojilkovic and Catt, 1992). These cells exhibit spontaneous action potential firing and express multiple types of Ca²⁺-mobilizing receptors. For example, TRH receptors in both cell types are coupled to the phospholipase C signaling pathway through G_q/G₁₁-proteins (Yu et al., 1998). Activation of this pathway leads to inositol trisphosphate-induced Ca²⁺ mobilization and sustained Ca²⁺ influx through voltage-gated and/or capacitative Ca²⁺ entry pathways. Facilitation of VGCI by TRH-induced Ca²⁺ mobilization has been attributed to membrane depolarization in response to inhibition of K⁺ current(s) (Sankaranarayanan and Simasko, 1996a; Schafer et al., 1999).

Calcium-mobilizing endothelin (ET) receptors are also expressed in several pituitary cell types (Stojilkovic and Catt, 1996), and their activation in lactotrophs stimulates prolactin (PRL) release (Kanyicska et al., 1995; Lachowicz et al., 1997). In contrast to TRH action, the ET-1-induced transient increase in PRL secretion is followed by a prolonged inhibition below basal levels. This sustained inhibitory action was initially observed by Samson et al. (1990) and Kanyicska et al. (1991), who also indicated the involvement of the ET_A receptor subtype in mediating this action (Samson, 1992; Kanyicska and Freeman, 1993). In parallel to the secretory profiles, activation of ET_A receptors stimulates a transient spike in [Ca²⁺]_i due to Ca²⁺ mobilization, followed by a sustained inhibition in response to the uncoupling of Ca²⁺ mobilization and entry pathways (Lachowicz et al., 1997). Such a bidirectional effect of a calcium-mobilizing agonist on Ca²⁺ signaling and secretion is unique among endocrine and neuroendocrine cells expressing G_q/G11-coupled receptors. Moreover, the cellular mechanisms mediating the sustained inhibition of PRL secretion and [Ca²⁺]_i are not known. Although it has been found that ET-1 activates Ca²⁺-sensitive K⁺ channels (I_K-Ca) (Kanyicska et al., 1997), this probably accounts only for the rapid hyperpolarization of cells during the transient rise in [Ca²⁺]_i, but not the sustained hyperpolarization when the [Ca²⁺]_i is well below basal levels. To investigate the cellular mechanisms underlying the sustained inhibition in PRL secretion, we used enriched lactotrophs, as well as GH₃ cells in which ET_A receptors have been transiently expressed. Cells were stimulated with ET-1, a common agonist for ET_A and ET_B receptors (Rubanyi and Polokoff, 1994). Our results indicate that the prolonged uncoupling between Ca²⁺ mobilization and VGCI pathways is due to activation of inward rectifier K⁺ (K_ir) channels via PTX-sensitive G-proteins.

	Materials and Methods

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Cell Cultures and Treatments. Experiments were performed on anterior pituitary cells from normal postpubertal female Sprague-Dawley rats obtained from Taconic Farms (Germantown, NY) and GH₃ immortalized pituitary cells. Pituitary cells were dispersed as described previously (Koshimizu et al., 2000) and cultured as mixed cells or enriched lactotrophs at a density of 10⁶ cells/25-mm coverslip in medium 199 containing Earle's salts, sodium bicarbonate, 10% heat-inactivated horse serum, and antibiotics. A two-stage Percoll discontinuous density gradient procedure (Koshimizu et al., 2000) was used to obtain an enriched lactotroph population. In single-cell measurements, lactotrophs were further identified by the addition of TRH.

Intracellular Calcium Measurements. [Ca²⁺]_i measurements were performed by imaging of fura-2-loaded cells. Cells attached to coverslips were immersed in 2 µM fura-2/acetoxymethyl ester (Molecular Probes, Eugene, OR) in medium 199 with Hanks' salts at 37°C for 1 h. The medium was changed to Krebs-Ringer buffer, and cells were kept in this medium at room temperature throughout the experiment. The samples were excited by alternating 334- and 380-nm light beams, and the emitted fluorescence was measured at 520 nm using an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) and an Attofluor imaging system (Atto Instruments, Rockville, MD). The ratio of the two intensities (F₃₄₀/F₃₈₀), which reflects the changes in [Ca²⁺]_i, was monitored in up to 75 cells simultaneously.

Electrophysiological Measurements. Current- and voltage-clamp recordings were performed at room temperature using an Axopatch 200 B patch-clamp amplifier (Axon Instruments, Union City, CA) and were low-pass-filtered at 2 kHz. Membrane potential (V_m) and current were measured using the perforated-patch recording technique (Rae et al., 1991). Briefly, amphotericin B (Sigma-Aldrich, St. Louis, MO) stock solutions (60 mg/ml) were prepared in dimethyl sulfoxide and stored for up to 1 week at 20°C. Just before use, the stock solution was diluted in pipette solution and sonicated for 30 s to yield a final amphotericin B concentration of 240 µg/ml. Patch electrodes used for perforated-patch recordings were fabricated from borosilicate glass (1.5 mm o.d.; World Precision Instruments, Sarasota, FL) using a Flaming Brown horizontal puller (P-87; Sutter Instruments, Novato, CA). Electrodes were heat polished to a final tip resistance of 3 to 6 M and then coated with Sylgard (Dow Corning Corporation, Midland, MI) to reduce pipette capacitance. Pipette tips were briefly immersed in amphotericin B-free solution and then backfilled with the amphotericin B-containing solution. A series resistance of <15 M was reached 10 min after the formation of a gigaohm seal (seal resistance >5 G) and remained stable for up to 1 h. When necessary, series resistance compensation was optimized. Pulse generation, data acquisition, and analysis were done with a personal computer equipped with a Digidata 1200 A/D interface in conjunction with Clampex 8 (Axon Instruments). The extracellular medium contained 120 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 4.7 mM KCl, 0.7 mM MgSO₄, 10 mM glucose, and 10 mM HEPES (pH adjusted to 7.4 with NaOH), and the pipette solution contained 50 mM KCl, 90 mM K⁺-aspartate, 1 mM MgCl₂, and 10 mM HEPES (pH adjusted to 7.2 with KOH). The bath contained <500 µl of saline and was continuously perfused at a rate of 2 ml/min using a gravity-driven perfusion system.

Simultaneous Recording of [Ca²⁺]_i and V_m. Pituitary 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 acetoxymethyl ester (Molecular Probes). The V_m was recorded as described above, and [Ca²⁺]_i was simultaneously monitored using a Nikon photon counter system as described previously (Van Goor et al., 2001b). The membrane potential and [Ca²⁺]_i were captured simultaneously at a rate of 5 kHz using a personal computer equipped with a Digidata 1200 A/D interface in conjunction with Clampex 8 (Axon Instruments). The [Ca²⁺]_i was calibrated in vivo according to the method of Kao (1994), and the values for R_min, R_max, S_f,480/S_b,480, and K_d were determined to be 0.75, 3.40, 2.45, and 230 nM, respectively.

Reverse transcription-PCR (RT-PCR) Analysis of K_ir 3.0 Isoform mRNA Expression. Total RNA from rat mixed pituitary cells, enriched lactotroph, or GH₃ cells was extracted using TRIzol reagent (Invitrogen). After DNase digestion, 2 µg of total RNA was reverse-transcribed into first-strand cDNA with oligo-dT primers and SuperscriptII reverse transcriptase (Invitrogen). The cDNAs were then amplified with different K_ir 3.0 isoform-specific primer sets in the nonconserved carboxyl-terminal region. The oligonucleotide sequences of primers used for PCR amplification, with accession numbers of the GenBank database given in parentheses, are listed as K_ir 3.1 (NM_031610): sense primer (1051-1072 bp, 5'-GCAACCTTTGAAGTCCCCACCC-3'), antisense primer (1435-1457 bp, 5'-GTAGGTCCTCCAGCCATCTTTTG-3'); K_ir 3.2 (NM_013192): sense primer (1335-1354 bp, 5'-TGGCTAACCGGGCAGAGCTG-3'), antisense primer (1477-1499 bp, 5'-CAGCTGAGGTCTACACTTTGGAC-3'); K_ir 3.3 (L77929): sense primer (1258-1275 bp, 5'-GGGAACTGGCAGAAGCTG-3'), antisense primer (1442-465 bp, 5'-CTGGTCTGCCACAGGGGGTTGTAG-3'); K_ir 3.4 (NM_017297): sense primer (1198-1220 bp, 5'-GGAAAAGGGCTTCTATGAGGTGG-3'), antisense primer (1437-1461 bp, 5'-CTCTGGGTAGCACACAGGCTTCACA-3').

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Uncoupling of Ca²⁺ Mobilization and Influx Pathways. About 60% of identified lactotrophs exhibited spontaneous fluctuations in [Ca²⁺]_i (Fig. 1A, left trace), whereas the residual cells were quiescent (Fig. 1B, left trace). The actions of ET-1 on Ca²⁺ signaling were studied in both spontaneously active and quiescent cells. In spontaneously active lactotrophs, addition of 100 nM ET-1 evoked a spike in [Ca²⁺]_i, which usually had a higher amplitude and longer duration than the spontaneously generated [Ca²⁺]_i transients. The spike phase was followed by the abolition of the [Ca²⁺]_i transients and a decrease in [Ca²⁺]_i below the initial level (Fig. 1A, left trace). The averaged basal [Ca²⁺]_i measured before and during the sustained stimulation with 100 nM ET-1 was significantly different (Fig. 1A, left bottom). In quiescent cells, 100 nM ET-1 induced a monophasic and extracellular Ca²⁺-independent rise in [Ca²⁺]_i (Fig. 1B, left trace). The amplitude and duration of the ET-1-induced spikes were comparable in quiescent and spontaneously active cells. Furthermore, depletion of extracellular Ca²⁺ abolished spontaneous [Ca²⁺]_i transients but did not affect ET-1-induced spike response (not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Typical patterns of ET-1-induced [Ca2+]i responses in lactotrophs (left) and GH3 cells expressing rat ETA receptors (right). A and B, representative traces in spontaneously active (A) and quiescent (B) cells. The numbers above traces indicate the fraction of spontaneously active or quiescent cells responding to ET-1with representative patterns. A, bottom, mean values ± S.E. of the [Ca2+]i estimated before and during the sustained ET-1 stimulation (shown in gray areas). Asterisks indicate a significant (P < 0.05) difference among means. In this and the following figures, calcium recordings were done in fura-2-loaded cells, and [Ca2+]i was expressed as the F340/F380 ratio, if not specified otherwise.

Role of VGCCs in ET-1-Induced Uncoupling. To study the dependence of ET-1-induced [Ca²⁺]_i responses in quiescent lactotrophs on the status of VGCI, we depolarized cells using Bay K 8644, an L-type Ca²⁺ channel agonist, and high concentrations of K⁺. Bay K 8644 (1 µM) initiated [Ca²⁺]_i transients in quiescent lactotrophs, as well as in somatotrophs and gonadotrophs (Fig. 2A) and several other unidentified cell types (not shown). These [Ca²⁺]_i transients in lactotrophs were inhibited by ET-1 in the same manner as in spontaneously active lactotrophs. Inhibition was also observed in somatotrophs, but not in gonadotrophs (Fig. 2A), indicating the cell type-specific action of ET-1 on inhibition of VGCI. In parallel with [Ca²⁺]_i signaling, in perfused pituitary cells ET-1 induced a transient stimulation of PRL release, followed by sustained inhibition below the basal level (Fig. 2B, left). An elevation in PRL secretion induced by 1 µM Bay K 8644 was also inhibited by ET-1 in the same manner (Fig. 2B, right). During 3-h incubation of pituitary cells in static culture, ET-1 inhibited basal and Bay K 8644 (1 µM)-stimulated PRL release with comparable IC₅₀ values in a picomolar concentration range (Fig. 2C).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Effects of ET-1 on Bay K 8644-induced calcium signaling and PRL release in pituitary cells. A, cell type-specific action of ET-1 on Bay K 8644-induced [Ca2+]i transients; typical traces are shown. B, profiles of ET-1-induced PRL secretion in perfused cells; representative records from control cells (left) and Bay K 8644-treated cells (right) are shown. ET-1 was added in the presence of Bay K 8644. C, concentration-dependent effect of ET-1 on PRL release in static cultures of control cells (left) and Bay K 8644-stimulated cells (right). Data points are means ± S.E. from sextuplicate incubations.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Depolarization-induced [Ca2+]i response in ET-1-stimulated and control lactotrophs. A, [Ca2+]i signaling in cells depolarized with 50 mM K+ and subsequently stimulated with ET-1. Data points are means derived from 32 control and 25 ET-1-stimulated cell records. B, representative traces of 25 mM K+-induced [Ca2+]i response in 100 nM ET-1-treated (left trace) and control cells (right trace). C, averaged data (means ± S.E.) of peak K+-induced [Ca2+]i responses from at least 25 cells per dose.

Signaling Pathway Mediating ET-1-Induced Uncoupling. Simultaneous measurements of V_m and [Ca²⁺]_i in lactotrophs revealed the existence of bursting V_m oscillations that gave rise to [Ca²⁺]_i transients similar to those observed in unclamped cells (Figs. 4A). Addition of 100 nM ET-1 evoked a rapid and prolonged hyperpolarization that terminated action potential firing and decreased the [Ca²⁺]_i (Fig. 4A). To test whether the ET-1-induced membrane hyperpolarization was caused by inhibition of a depolarizing current or activation of a hyperpolarizing current, the input resistance of the membrane was monitored by the application of hyperpolarizing current injections. In all lactotrophs examined (n = 5), ET-1 decreased the cell input resistance (Fig. 4B), indicating the activation of a hyperpolarizing current. Consistent with this, ET-1 increased the amplitude of a current evoked by hyperpolarizing step from 70 mV to 150 mV (Fig. 4C). The ET-1-induced current was observed in 90 to 170 mV voltage range and reversed its direction at about 80 mV (Fig. 4D), the reversal potential for K⁺ in these experimental conditions. The current was masked in potentials positive to 70 mV due to activation of other channels (not shown). These results suggest that ET-1 activates a K⁺ current, leading to hyperpolarization of cells and abolition of action potential firing.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   ET-1-induced membrane hyperpolarization and activation of a K+ current. A, simultaneous measurement of Vm and [Ca2+]i in response to 100 nM ET-1. B, ET-1-induced reduction in membrane input resistance (upper trace), monitored by injecting regular hyperpolarizing current pulses (lower trace). C, activation of an inward current by a hyperpolarizing voltage step from 70 mV to 150 mV before and during application of 100 nM ET-1. D, current-voltage relation of ET-1-activated current before () and during () application of 100 nM ET-1. The traces shown in A and C are from the same cell.  and  in A indicate the time when currents shown in C and D were measured. Data points in D are mean ± S.E. from five experiments. In this figure and Figs. 6 and 8, the current activated by hyperpolarizing pulses was recorded in perforated patch-clamp conditions with 4.7 mM extracellular K+. Vcmd, the command voltage. Calcium recordings were done in indo-1-loaded cells, and [Ca2+]i was calculated as described under Materials and Methods.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effects of PTX (250 ng/ml overnight) on ET-1-induced calcium signaling in lactotrophs and GH3 cells transfected with rat ETA receptors. ET-1-induced [Ca2+]i response in spontaneously active (A) and quiescent (B) cells. The tracings shown are representative, with numbers above traces indicating the fraction of spontaneously active or quiescent cells responding to ET-1 with representative patterns. A, bottom, mean values ± S.E. of [Ca2+]i estimated before and during the sustained ET-1 stimulation (shown by gray areas). Asterisks indicate significant (P < 0.05) differences among means. C, effects of nifedipine on sustained ET-1-induced VGCI; representative traces are shown.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   ET-1-induced membrane depolarization in PTX-treated (250 ng/ml overnight) lactotrophs. A, simultaneous measurement of Vm and [Ca2+]i in response to 100 nM ET-1 (bar). B, abolition of the ET-1-induced reduction in input resistance by PTX treatment. C, the lack of ET-1-induced activation of K+ current in PTX-treated cells. D, current-voltage relation of K+ current before () and during () application of 100 nM ET-1. The traces shown in A and C are from the same cell.  and  in A indicate the time when currents shown in C and D were measured. Data points in D are mean ± S.E.M. from five experiments. Calcium recordings were done in indo-1-loaded cells.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   The lack of effects of forskolin (A) and 8-Br-cAMP (B) on Ca2+ signaling in ET-1-stimulated lactotrophs. Left, representative patterns of Ca2+ signaling in spontaneously active lactotrophs. Center, comparison of ET-1- and 100 nM TRH-induced [Ca2+]i responses in quiescent lactotrophs. Right, mean values ± S.E. of [Ca2+]i in spontaneously active lactotrophs estimated during the first 3 min (basal) and between 6 and 9 min of recording (ET-1-treated). Asterisks indicate significant (P < 0.05) differences among means.

Characterization of K⁺ Channels Involved in ET-1-Induced Uncoupling. In accordance with literature data in other pituitary cell types (Kuryshev et al., 1997), 5 mM Cs⁺ reduced the K⁺ current in lactotrophs (Fig. 8, A and B). Single-cell [Ca²⁺]_i measurements also revealed that the addition of 5 mM Cs⁺ initiated [Ca²⁺]_i transients in a fraction of quiescent lactotrophs (33%) and increased the frequency of transients in spontaneously active cells (80%; Fig. 8C). In the presence of Cs⁺, the ET-1-induced spike [Ca²⁺]_i response was followed by a sustained plateau response in a large fraction of lactotrophs. The sustained plateau response was frequently observed in lactotrophs in which Cs⁺ initiated [Ca²⁺]_i transients (Fig. 9A, left trace), as well as in cells in which Cs⁺ per se was ineffective (Fig. 9A, right trace). These results indicate that a Cs⁺-sensitive current is constitutively active in lactotrophs and contributes to the control of spontaneous action potential firing, as well as to uncoupling of Ca²⁺ mobilization and influx pathways in ET-1-stimulated lactotrophs.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Effects of extracellular Cs+ on K+ current and [Ca2+]i signaling in lactotrophs. A and B, activation of K+ current by a hyperpolarizing voltage step from 60 mV to 140 mV before and during application of 5 mM Cs+. A, representative traces. B, current-voltage relation. C, effects of 5 mM Cs+ on [Ca2+]i in quiescent (left trace) and spontaneously active cells (center and right trace).

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Effects of K+ channel blockers on [Ca2+]i signaling in 100 nM ET-1-stimulated lactotrophs. A, pattern of ET-1-induced [Ca2+]i signaling in lactotrophs bathed in medium containing Cs+; representative traces in lactotrophs responding to the addition of 5 mM Cs+ with (left trace) and without (right trace) elevation in [Ca+]i are shown. B, pattern of ET-1-induced [Ca2+]i signaling in lactotrophs bathed in medium containing 1 µM E-4031, a specific erg-like channel blocker; representative traces are shown. The numbers above traces in A and B indicate the fraction of cells responding with a particular pattern to Cs+ + ET-1 and E-4031 + ET-1 stimulation. C, mean values ± S.E. of basal and sustained ET-1-induced [Ca2+]i responses in control cells, and 5 mM Cs+- and 1 µM E-4031-treated cells, with N indicated in parentheses. Asterisks indicate significant (P < 0.05) difference among means. D, the lack of effect of 100 nM apamine and 100 nM charybdotoxin cocktail on the pattern of ET-1-induced Ca2+ signaling in spontaneously active (left trace) and quiescent lactotrophs (right trace). Numbers above traces indicate the fraction of cells responding with a particular pattern.

View larger version (55K): [in this window] [in a new window]   Fig. 10.   Expression of Kir 3.0 channels in anterior pituitary cells and enriched lactotrophs. Black, detection of Kir 3.0 isoforms in mixed anterior pituitary cells (lanes 3 and 4) and purified lactotrophs (lanes 5 and 6). For negative control cells, PCR was conducted using first-strand cDNA samples without RT (lanes 3 and 5). In the case of "no template" control (lane 2), water was substituted for the first-strand cDNA sample in the PCR reaction. DNA markers are shown in lane 1. GAPDH primers were used as an internal control to monitor the quality of RNA preparation. Gray, Southern blot analysis of the same gels shown above. Blots were probed with 3'-end-labeled oligonucleotide specific to a corresponding type of Kir channel.

	Discussion

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Activation of Ca²⁺-mobilizing receptors in excitable cells usually generates biphasic [Ca²⁺]_i and secretory profiles composed of an early spike response mediated by inositol trisphosphate-induced Ca²⁺ release and a sustained plateau response that is dependent on Ca²⁺ influx through voltage-gated and -insensitive calcium channels (Stojilkovic and Catt, 1992). In lactotrophs, stimulation of Ca²⁺ mobilization and PRL secretion by ET-1 and their PTX insensitivity are consistent with the coupling of ET_A receptors to the G_q/11 signaling pathway and activation of the phospholipase C signaling pathway (Stojilkovic et al., 1990b, 1992). In contrast to other Ca²⁺-mobilizing receptors, the ET-1-induced Ca²⁺ mobilization in lactotrophs is not followed by sustained increase in Ca²⁺ influx due to the prolonged membrane hyperpolarization, which inhibits VGCI (Lachowicz et al., 1997). Here we show that in PTX- and Cs⁺-treated lactotrophs, ET-1 induced biphasic [Ca²⁺]_i and secretory responses comparable with those observed upon stimulation with other Ca²⁺-mobilizing agonists (Hinkle et al., 1996; Lachowicz et al., 1997). Thus, ET_A receptors in these cells also trigger a common mechanism for the activation of Ca²⁺ influx with Ca²⁺ mobilization, but the coupling of these receptors to the PTX-sensitive G_i/o signaling pathway under physiological conditions prevents the development of the second phase in Ca²⁺ signaling.

In other cell types, intracellular signaling through the G_i/o pathway is well characterized. During receptor activation, the  subunit of these heteromeric proteins couples to adenylyl cyclase, leading to inhibition of cAMP production, whereas / dimer activates several other effector molecules in a cell-specific manner, including phospholipase C, VGCCs, and K_ir channels (family 3.0) (Wickman and Clapham, 1995). As discussed above, the ET_A receptor signals for activation of phospholipase C through PTX-insensitive G proteins. Furthermore, the [Ca²⁺]_i response to the addition of extracellular K⁺ was comparable in control and ET-treated cells, suggesting that VGCCs are not directly inhibited. In accordance with this finding, activation of ET_A receptors in other pituitary cell types was found not to affect voltage-gated Ca²⁺ current (Tomic et al., 1999). Although in pituitary cells basal adenylyl cyclase activity is controlled by spontaneous VGCI (Kostic et al., 2001) and ET-1 inhibits it in a dose-dependent and PTX-sensitive manner (Tomic et al., 1999), it is unlikely that the adenylyl cyclase-signaling pathway mediates the action of ET_A receptors on inhibition of VGCI. First, activation of adenylyl cyclase by forskolin did not affect the pattern of ET-1-induced [Ca²⁺]_i signaling. Second, the addition of 8-Br-cAMP, a permeable cAMP analog, was also ineffective.

On the other hand, several lines of evidence support the role of K⁺ channels in preventing VGCI after intracellular Ca²⁺ mobilization. The sustained membrane hyperpolarization and decrease in input resistance observed in ET-1-stimulated cells indicate that ET_A receptors activate or augment a hyperpolarizing current rather than inhibit a depolarizing current. In voltage-clamped cells stimulated with ET-1, application of hyperpolarizing pulses revealed stimulation of a K⁺ current. Agonist-induced hyperpolarization of cells, reduction in input resistance, and activation of K⁺ current were abolished in PTX-treated cells. Addition of extracellular Cs⁺ inhibited hyperpolarization-induced K⁺ current. This treatment also led to the coupling of Ca²⁺ influx to Ca²⁺ mobilization in a manner comparable with that observed in PTX-treated cells, suggesting a role of Cs⁺-sensitive K⁺ channels in sustained hyperpolarization of cells.

Pituitary lactotrophs express two channel subtypes that are sensitive to Cs⁺ at the concentrations used in our experiments, K_ir and erg-like channels, but only the latter is also sensitive to E-4031 (Einhorn et al., 1991; Schafer et al., 1999; Schledermann et al., 2001). Initiation of [Ca²⁺]_i transients in quiescent lactotrophs and the increase in the frequency of [Ca²⁺]_i transients in spontaneously active cells after the addition of 5 mM Cs⁺ and 1 µM E-4031 are consistent with the role of erg-like channels in controlling the firing frequency in unstimulated cells. However, in cells treated with E-4031, ET-1-induced inhibition of sustained VGCI did not differ from that observed in control cells, supporting a role for K_ir rather than erg-like channels in mediating the ET-induced sustained V_m hyperpolarization. Consistent with these findings, our results indicate that lactotrophs express the K_ir 3.0 subfamily of channels. Others have also observed the expression of K_ir 3.0 in pituitary tissue and GH cells (Wulfsen et al., 2000; Gregerson et al., 2001). Further single-channel recordings are needed to fully characterize these channels and their regulation by ET_A receptors.

The cross-coupling of ET_A receptors to the G_i/o signaling pathway is not unique to pituitary lactotrophs, as it is also observed in other cell types (James et al., 1994; Ono et al., 1994). Other Ca²⁺-mobilizing receptors also cross-couple to the G_i/o signaling pathway (Krsmanovic et al., 1998). In lactotrophs, the pattern of ET-1 action depends on the firing status of individual cells. In quiescent cells, the ET-1-induced monophasic [Ca²⁺]_i response was solely dependent on Ca²⁺ mobilization, whereas in spontaneously active cells, the Ca²⁺-mobilizing action was accompanied by a prolonged inhibition of spontaneous pacemaking and VGCI. Because spontaneous action potential firing and the associated Ca²⁺ influx are sufficient to trigger PRL secretion (Sankaranarayanan and Simasko, 1996b; Van Goor et al., 2001a), the ET-induced hyperpolarization and the ensuing inhibition of VGCI are an effective mechanism for the sustained inhibition of PRL secretion. The similar actions of ET-1 in lactotrophs and GH cells, the latter not expressing K_ir 3.4, further indicate that this particular isoform of channels is not essential for ET-1-induced inhibition of VGCI.

The coupling of ET_A receptors to K_ir in spontaneously active cells and their ability to induce sustained membrane hyperpolarization and inhibition of hormone secretion are comparable with the action of dopamine and somatostatin receptor activation in pituitary lactotrophs and other cell types. Like ET_A receptors (Tomic et al., 1999), these receptors are negatively coupled to adenylyl cyclase; i.e., their activation leads to inhibition of cAMP production in a PTX-sensitive manner (Freeman et al., 2000). Dopamine and somatostatin also directly activate K_ir in a PTX-sensitive manner (Einhorn et al., 1991; Sims et al., 1991; Lledo et al., 1992). However, in contrast to ET_A receptors, dopamine and somatostatin receptors do not stimulate the phospholipase C in pituitary cells (Tallent et al., 1996; Freeman et al., 2000). Furthermore, dopamine and somatostatin inhibit L-type calcium channels (Lewis et al., 1986; Kleuss, 1995; Tallent et al., 1996), whereas ET_A receptors do not. At the present time, the biochemical reason for the difference in the coupling of these receptors to the L-type channels is not clear.

Despite the importance of K_ir channels in mediating the sustained membrane hyperpolarization, they are not exclusively responsible for ET-1-induced termination of action potential firing in lactotrophs. Both lactotrophs and GH₃ cells express I_K-Ca channels (Ritchie, 1987b; Van Goor et al., 2001b), and their activation by Ca²⁺ mobilization in response to ET-1 and TRH has been shown previously (Ritchie, 1987a; Kanyicska et al., 1997). Consistent with this, in PTX- and Cs⁺-treated cells, the sustained facilitation of VGCI often occurs with a delay, effectively dissociating the spike and plateau [Ca²⁺]_i responses. Under normal conditions, however, depletion of the intracellular Ca²⁺ pool(s) within 1 to 2 min and the marked reduction in [Ca²⁺]_i caused by inhibition of VGCI will prevent the sustained activation of I_K-Ca channels and, thus, their participation in facilitating membrane hyperpolarization.

In conclusion, our results indicate that ET-1 prevents VGCI after intracellular Ca²⁺ mobilization in lactotrophs by activating the K_ir 3.0 family of channels. The cross-coupling of ET_A receptors to the G_i/G_o signaling pathway leads to activation of these channels, hyperpolarization of cells, and abolition of spontaneous firing of action potentials and action potential-driven Ca²⁺ influx. Abolition of this cross-coupling by PTX unmasks the depolarizing action of ET-1, typically observed in response to other Ca²⁺-mobilizing receptors. The uncoupling of Ca²⁺ mobilization and influx pathways and the inhibition of spontaneous firing of action potentials are effective mechanisms for the prolonged inhibition of PRL secretion.