Introduction

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Antidiuretic hormone and OXT are the main hormonal secretory products of the neurohypophysis. ADH plays an integral role in the maintenance of fluid homeostasis and vascular tone; OXT is a neurohormonal mediator of various reproductive functions (Martin, 1986). It has long been known that dopamine modulates the release of these neuropeptides in vivo (Bridges et al., 1976; Moos and Richard, 1982; Melis et al., 1990). It is unclear, however, whether dopaminergic agents act directly on the neurohypophysis, or if they exert their action on neurons located more proximally within the integrative circuitry of the hypothalamus (Crowley et al., 1991). Direct electrophysiological study of the neurohypophysis should help clarify this issue.

Animal studies suggested that the stimulatory effect of apomorphine on neurohypophysial peptide release was mediated through a D₂ dopamine receptor (Amico et al., 1992). Although conflicting reports have since implicated other receptor subtypes, much of the available evidence still supports the initial claim that this effect is transduced through a dopaminergic receptor which has "D₂-like" pharmacology (Parker and Crowley, 1992; Uvnas-Moberg et al., 1995). Recently, molecular cloning has revealed that the D₂-like family of dopamine receptors consists of at least three distinct membrane proteins: D₂, D₃ and D₄ (Gingrich and Caron, 1993). Great effort currently is being made in the development of selective ligands for each of these receptor subtypes. Although several highly selective D₄ antagonists are now commercially available, no D₄ agonist has yet been described (Strange, 1994; Kebabian, et al., 1997).

Dopamine has been shown to modulate potassium channel function in a variety of neuronal preparations (Stack and Surprenant, 1991; Liu et al., 1996). In the past, the small size and inaccessibility of peptidergic nerve terminals has impeded progress in understanding the membrane events governing neurohypophysial hormone secretion. To overcome this problem, several approaches have been developed for making intracellular recordings in the posterior pituitary (Lemos and Nowycky, 1989; Bourque, 1990; Jackson et al., 1991). In this study we have used patch-clamp techniques to investigate the effect of dopamine receptor activation on I_K in voltage-clamped nerve terminals in neurohypophysial slices.

	Methods

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Slice preparation. Experiments were performed on tissue from Sprague-Dawley rats of either sex weighing 240 to 260 g. Animals were housed under constant 12/12 hr light/dark cycle with free access to water and food. After CO₂-induced narcosis and decapitation, the brain was removed and discarded. The cranium was then filled with ice-cold aCSF: 115 mM NaCl, 4.0 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 2 mM CaCl₂, 1 mM MgCl₂, and 10 mM glucose, saturated with 95% O₂/5% CO₂. The neurointermediate and anterior pituitary lobes were removed from the calvarium and separated by gentle insertion of fine forceps. The neurointermediate lobe was then glued to a plastic block, submerged in ice-cold saline and sliced with a Lancer Vibratome at a thickness of 60 to 75 µm. All slices were stored at room temperature (21-24°C) in 95% O₂/5% CO₂-saturated aCSF and used within 3 hr (Jackson et al., 1991; Jackson, 1993).

Patch-clamp electrophysiology. Patch-clamp recordings were made with an EPC-9 patch-clamp amplifier interfaced to a MacIntosh computer. Stimulus and data acquisition were carried out with the computer program Pulse (Instrutech, Great Neck, NY). Tissue slices were perfused with aCSF (as above), bubbled with 95% O₂/5% CO₂ at room temperature at a rate of 2 to 4 ml/min through a simple gravity feed system. Individual nerve terminals at the slice surface were located with an upright Nomarski microscope (Reichert Jung diastar) and a Zeiss 40× water immersion, long working distance objective (Jackson et al., 1991; Jackson, 1993). Patch pipettes were fabricated from thin-walled borosilicate glass, and the pipette shanks were coated with Sylgard to reduce electrode capacitance (Hamill et al., 1981).

Drug application. All dopaminergic agents used in these experiments were obtained from Research Biochemicals International (Natick, MA). These compounds were dissolved in aCSF and applied to the preparation by superfusion. Before the addition of drugs, current was recorded at 15- or 30-sec intervals for 1 to 3 min to verify the stability of the baseline. Current was also recorded after the removal of any drug(s) to demonstrate viability of the cell and recovery of I_K to the original baseline. In experiments conducted with highly lipophilic drugs like PPHT, the agent was first dissolved in dimethyl sulfoxide, then diluted into aCSF to obtain the desired final drug concentration. The final concentration never exceeded 0.1% dimethyl sulfoxide; this vehicle, without drug, was tested and had no effect on whole-terminal I_K.

Data analysis. Current records were analyzed on a MacIntosh computer with the computer program PulseFit (Instrutech Inc., Great Neck, NY). This program was used to fit current decays to a double-exponential function. The computer program Origin (MicroCal, Northampton, MA) was used on a personal computer to fit data to a Boltzmann function. Simple statistical analyses were performed on exported data with the program Microsoft Excel. When arithmetic means were computed, they were presented with the standard error of the mean. All null hypotheses were subjected to the appropriate t test, and a level of P < .05 was considered statistically significant.

	Results

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Inhibition of outward current. Consistent with previous studies on this preparation (Jackson et al., 1991; Bielefeldt et al., 1992), all nerve terminals in the current study displayed a prominent outward current in response to depolarizing test pulses under voltage clamp (n > 50). Pharmacological and biophysical studies have established that this is an I_K (Bielefeldt et al., 1992). Voltage steps from 100 mV to +10 mV rapidly activated this current (fig. 1), which subsequently inactivated while the test potential was held constant.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Outward K+ current in voltage-clamped neurohypophysial nerve terminals. After a 250-msec prepulse at 100 mV, voltage was stepped to +10 mV for 500 msec, then returned to the holding membrane potential of 80 mV. In this representative set of tracings, a brief (<5 msec) tetrodotoxin-sensitive inward Na+ current can be seen at the beginning of each depolarizing test pulse. A prominent outward current is rapidly activated and then decays over time. When the same nerve terminal was depolarized after 5 min of exposure to 30 µM PPHT, a marked reduction in both peak and final K+ current was observed.

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View larger version (18K): [in this window] [in a new window]   Fig. 2.   Reversibility of the PPHT effect. IK was recorded at 30-sec intervals by the stimulation paradigm described in figure 1. Superfusion with 10 µM PPHT (indicated by the bar) reduced both peak and final components of this outward K+ current, and the effect was completely reversed when the agonist was washed from the medium. Final current represents the mean current calculated during the last 200 msec of each 500-msec depolarizing pulse (as shown in fig. 1).

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View larger version (11K): [in this window] [in a new window]   Fig. 3.   PPHT concentration-response curve. At each concentration, outward K+ current was recorded before and after a 5-min superfusion with the dopamine receptor agonist, PPHT. The stimulation paradigm for each voltage pulse was identical with that described in figures 1 and 2. Final current in the presence of PPHT was normalized to the predrug control response for the same individual nerve terminal. Each concentration point is a mean ± S.E.M. for three to six nerve terminals. The best curve fit (see text for equation and parameters) describing this concentration-response relationship is shown as a dotted line.

Dopamine-receptor specificity. Preincubation of nerve terminals with 100 µM eticlopride, a nonselective antagonist for D₂-like dopamine receptors (D₂, D₃ and D₄), completely eliminated the inhibitory effect of 10 µM PPHT (fig. 4). By itself, 100 µM eticlopride had no effect on nerve terminal I_K. To address the issue of dopamine receptor subtype, we tested neurohypophysial nerve terminal I_K with a panel of dopaminergic agonists (fig. 5). The agonist, SKF 38393, binds to members of the "D₁-like" receptor family (D_1A and D_1B/D₅) with an apparent affinity in the nanomolar range (Madras et al., 1990); yet 100 µM SKF had no effect on I_K in our preparation.

View larger version (66K): [in this window] [in a new window]   Fig. 4.   Elimination of the PPHT effect with a D2-like receptor antagonist. Final IK was assessed by the same pulse protocol used in figure 1. After establishing a stable current baseline (3-5 min), nerve terminals were superfused with either aCSF alone or with aCSF containing 100 µM eticlopride for a full 5 min before the addition of 10 µM PPHT. This concentration of agonist (PPHT) was chosen based on its location along the concentration-response curve just above the inflection point (fig. 3). Data represent the mean ± S.E.M. for three to six individual nerve terminals. * indicates P < .05 between columns.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   D4-type dopamine receptor selectivity. Final neurohypophysial K+ current was monitored in the presence of various dopamine receptor agonists, according to the same pulse paradigm used in figure 1. After establishing a stable base line, nerve terminals were superfused for 5 min with either SKF 38393 (D1/D5 selective), quinpirole (D2/D3 selective) or U101958 (D4 selective), and then reassessed. All agonists were presented at 100 µM. Final K+ current was normalized to the predrug control response for each individual nerve terminal and averaged. Data represent the mean ± S.E.M. for three to six individual nerve terminals. The * indicates that the effect of U101958 was significantly different from that of SKF 38393 and quinpirole (P < .05).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Reversible reduction of IK by U101958. IK was recorded at 15-sec intervals with the pulse paradigm used in figure 1. The time of U101958 superfusion is indicated by the bar above. Like PPHT, 100 µM U101958 reduced both peak and final components of IK. The effect was reversed when agonist was washed from the medium.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Blockade of the PPHT response by a D4-type dopamine receptor antagonist. IK was recorded at 15-sec intervals before, during and after challenge with agonist (pulse paradigm as in fig. 1). Both peak and final components of IK were reversibly inhibited by 10 µM PPHT (closed symbols). When a D4-selective antagonist (10 µM RBI-257) was added to the medium, the action of PPHT was blocked (open symbols). Data represent the mean of three control experiments and four blockade experiments. See text for mean normalized current values.

Kinetic characterization. To investigate the mechanism of this alteration in neurohypophysial I_K, we studied the effect of D₄ receptor activation on the voltage dependence of K⁺ channel activation. Outward current was recorded before and after a 5-min exposure to 30 µM PPHT. By use of test pulses varying from 70 to 30 mV, current-voltage relationships were constructed for both peak and final I_K (fig. 8). At all voltages tested PPHT reduced I_K by approximately proportional amounts, which indicates that D₄ receptor activation does not produce its inhibitory effect by shifting the voltage dependence of these K⁺ channels.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Current plotted versus voltage for peak (triangles) and final (squares) K+ current. Voltage-clamped nerve terminals were held at 80 mV and interrupted at 5-sec intervals with 500-msec pulses to each of the depolarizing voltages shown (open symbols). After a 5-min exposure to 30 µM PPHT (closed symbols), the same nerve terminals were again subjected to a repeat series of voltage pulses from 70 through 30 mV. Data points (connected by solid lines) represent the mean current for six individual terminals.

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View larger version (13K): [in this window] [in a new window]   Fig. 9.   Current inactivation profile. Voltage-clamped nerve terminals were held at various prepulse potentials (from 130 to 40 mV) for 500 msec, then stepped to +10 mV for an additional 500 msec. Peak current at 10 mV was then plotted as a function of prepulse voltage (open circles) to assess the voltage-dependent inactivation of these neurohypophysial K+ channels. After a 5-min exposure to 30 µM PPHT, each individual terminal was subjected to the same test series, and peak current was again plotted against prepulse voltage (closed circles). Data represent the mean values from six experiments. Best fitting Boltzmann functions are drawn as dotted lines. PPHT did not alter the voltage dependence of inactivation (see text for parameter values).

	Discussion

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This study has shown that activation of a dopamine receptor inhibits I_K in the neurohypophysis. The nerve terminals of this preparation previously have been shown to contain three different types of K⁺ channels (Bielefeldt et al., 1992). One type conducts a rapidly inactivating transient outward current, I_A, which shares many common properties with the transient outward current of the cell bodies within the hypothalamic nuclei from which these axons originate (Cobbett et al., 1989). A second type, I_BK, is a Ca⁺⁺-dependent K⁺ channel which differs in its kinetic profile from hypothalamic Ca⁺⁺-dependent K⁺ channels. The data obtained from our kinetic analysis indicate that these two channel types are functionally altered by D₄ receptor activation. The third type of neurohypophysial K⁺ channel, the D channel, is slowly activating and shows no inactivation (Bielefeldt et al., 1992). Because of the small and variable contribution made by the D channel to whole terminal I_K, we cannot say whether I_D is also modulated by dopamine receptor activation.

Activation of dopamine receptors expressed in a neuronal cell line increased conductance through inwardly rectifying K⁺ channels (Greif et al., 1995). To our knowledge, no inwardly rectifying K⁺ channels have been observed within the nerve terminals of the neurohypophysis. Furthermore, the dopamine response described here is an inhibition of I_K rather than an enhancement. Voltage-clamp studies in rat hippocampal pyramidal neurons (Pedarzani and Storm, 1995) have demonstrated a similar dopamine receptor-mediated reduction in a slow Ca⁺⁺-dependent K⁺ current (I_AHP). This is similar to our finding a reduction in a Ca⁺⁺-dependent K⁺ current (I_BK). However, in the neurohypophysis another I_K, the A-current, was also reduced. The observation that two distinct K⁺ channels can be modulated by the same dopamine receptor is not without precedent. With a mesencephalic neuronal preparation derived from embryonic rats, Liu et al. (1994) found that quinpirole is capable of simultaneously altering both an I_A and a delayed rectifier through a common G-protein-mediated mechanism.

A decade ago, it was widely accepted that dopamine acted through two receptor subtypes: D₁ and D₂ (Sokoloff and Schwartz, 1995). In this schema based on pharmacological data, butyrophenone antipsychotics (such as haloperidol) bound the D₁ receptor with very low affinity and the D₂ receptor with high affinity. The recent cloning and sequencing of dopamine receptors has changed this view considerably. These studies have identified six unique dopamine receptor subtypes (Sibley et al., 1993; Gingrich and Caron, 1993). However, these six are in two discrete families, now called "D₁-like" and "D₂-like." Members of the D₁-like family (D_1A, D_1B and D₅ receptors) share a very high degree of sequence homology within their transmembrane domains, and they have only limited distribution within the rat central nervous system (Gingrich and Caron, 1993). The D₁ agonist, SKF 38393, binds all three members of this D₁-like receptor family with relatively high affinity. Our finding that 100 µM SKF38393 had no effect on potassium current in peptidergic nerve terminals indicates that a D₁-like receptor is not involved in this response.

Members of the D₂-like family of dopamine receptors (D₂, D₃ and D₄) exhibit a considerable amount of overlap in their ability to bind various ligands. The nonselective D₂-like dopamine receptor agonist, PPHT, reversibly inhibited neurohypophysial I_K in a concentration-dependent fashion. Furthermore, the D₂-like dopamine receptor antagonist eticlopride blocked the action of PPHT. These results indicate that the receptor responsible for the reduction in neurohypophysial I_K is a member of the D₂-like dopamine receptor family. Because the PPHT response can be blocked by the highly selective D₄ dopamine receptor antagonist, RBI-257, it is likely that the PPHT response is mediated by this receptor. Quinpirole, a ligand known to exhibit nanomolar affinity at both the D₂ and D₃ dopamine receptors (Sokoloff et al., 1990; Levesque et al., 1992; Freedman et al., 1994) and to activate these receptors at concentrations smaller than 100 µM (Liu et al., 1996), had no effect on neurohypophysial I_K at this concentration. A caveat in our identification of this receptor as a D₄ subtype is the report of quinpirole activation of the human receptor (Ten Brink et al., 1996). Nevertheless, our results suggest that the only dopamine receptor with an effect on K⁺ current in our preparation is the D₄ subtype. The present study represents the first report of an in situ response transduced by a D₄ dopamine receptor. The reduction of a K⁺ current described here is similar to that seen in cells stably transfected with D₄ receptor cDNA (Colville et al., 1994)

We also investigated the action of U101958, a compound that binds to D₄ dopamine receptors with an apparent affinity in the nanomolar range (Schlachter et al., 1995; Kebabian et al., 1997); its affinity for D₂ and D₃ receptors is approximately 1000-fold less (Kebabian et al., 1997). This compound was shown to antagonize responses mediated by a human D₄ receptor (TenBrink et al., 1996). Our observation that U101958 reduces neurohypophysial I_K demonstrates that this ligand acts as an agonist in the rat posterior pituitary, and a D₄-receptor-specific agonist should be a useful experimental tool in probing the function of this receptor subtype.

The observation that D₄ receptor activation alters I_K within the neurohypophysis has important implications regarding the dopaminergic modulation of neuropeptide release in vivo. An increase in the frequency of neurohypophysial impulses enhances neuropeptide release (Gainer et al., 1986; Armstrong et al., 1989), presumably through the sequence of partial I_K inactivation, action potential broadening and increased calcium entry (Gainer et al., 1986; Bourque, 1990; Jackson et al., 1991). This would suggest that D₄ receptor activation, with its inhibitory effect on presynaptic I_K, could enhance neurohypophysial peptide release in a similar fashion.

The ability to secrete ADH or OXT in response to an appropriate stimulus depends on the neurohypophysis receiving information from various sensor elements. The release of ADH is affected by small (<1%) variations in plasma osmolality, a signal which is detected and integrated through hypothalamic osmoreceptors (Oliet and Bourque, 1994). Although neurohypophysial cell bodies have been shown to generate action potentials in vitro after the application of hyperosmotic solutions (Oliet and Bourque, 1993), many investigators believe that these neurons are at least one synapse away from the actual osmoreceptors (Reeves and Andreoli, 1992; Oliet and Bourque, 1994). In a recently published whole-animal study, intracerebroventricular injection of the D₂-like dopamine receptor antagonist, haloperidol, was shown to attenuate the increase in circulating levels of ADH induced by intravenous infusion of hypertonic saline (Yamaguchi et al., 1996). It is possible, therefore, that dopaminergic neurotransmission within the neurohypophysis contributes to the regulation of the ADH secretory response to an osmotic load in vivo. The D₄-receptor-mediated inhibition of neurohypophysial I_K described above provides at least one potential mechanism whereby endogenous dopamine may mediate such an effect.