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1Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; 2Department of Bioengineering, University of California, San Diego, La Jolla, California; and 3Department of Physiology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada
Submitted 18 January 2005; accepted in final form 14 March 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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50%. This effect on ICa(L) is mimicked by dialyzing a Gi-activator peptide (10–7 M) into these cells, implicating a role for the inhibitory G protein, Gi. These NPR-C–mediated effects were specific to ICa(L). T-type Ca2+ channels were unaffected by CNP. Current-clamp experiments revealed the ability of CNP, acting via the NPR-C receptor, to decrease (25%) the number of action potentials elicited during a 500 ms depolarizing stimulus. Analysis of action potential duration revealed that CNP and cANF significantly decreased 50% repolarization time (APD50) in MNCs. In summary, our findings show that CNP has a potent and selective inhibitory effect on ICa(L) and on excitability in MNCs that is mediated by the NPR-C receptor. These data represent the first electrophysiological evidence of a functional role for the NPR-C receptor in the mammalian hypothalamus. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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), natriuretic peptides have been shown to act as essential regulators of fluid and electrolyte homeostasis in the mammalian CNS (Levin et al. 1998). While three distinct natriuretic peptides, including ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), have been isolated, it is CNP that is most abundant in the mammalian brain (Herman et al. 1993; Kaneko et al. 1993; Ogawa et al. 1992; Yamamoto et al. 1997). Measurements of immunoreactivity in the area of the hypothalamus indicate CNP expression levels >50 times greater than those of ANP or BNP (Herman et al. 1993; Imura et al. 1992). Furthermore, injection of CNP into the third ventricle of the rat brain potently inhibits the release of vasopressin (VP) (Samson et al. 1991; Shirakami et al. 1993; Yamamoto et al. 1991). This effect is two orders of magnitude more potent than that of either ANP or BNP (Yamamoto et al. 1991, 1997), indicating that CNP is the critical natriuretic peptide modulating MNC excitability.
CNP binds with high affinity to two receptors which have been denoted natriuretic peptide receptor types B and C (NPR-B and NPR-C) (Levin et al. 1998). The NPR-B receptor includes an intracellular particulate guanylyl cyclase domain that mediates an increase in intracellular cGMP levels when CNP is bound to it (Lucas et al. 2000). The NPR-C receptor does not contain a guanylyl cyclase domain and does not alter cGMP levels directly (Levin et al. 1998; Lucas et al. 2000; Maack 1992). In the absence of any evidence to the contrary, the inhibitory effect of CNP on hormone release from MNCs in the hypothalamus has been attributed to the NPR-B receptor and a subsequent increase in intracellular cGMP; however, this putative mechanism has not been tested directly (Shirakami et al. 1993; Yamamoto et al. 1997).
NPR-C is prominently expressed in the mammalian hypothalamus (Peng et al. 1996; Sumners and Tang 1992), but it has been largely assigned the role of a physiologically silent receptor in the CNS (Imura et al. 1992). In the heart and in gastrointestinal smooth muscle, NPR-C has been functionally linked to adenylyl cyclase via a pertussis toxin-sensitive G protein (Anand-Srivastava and Cantin 1986; Anand-Srivastava et al. 1996; Murthy and Makhlouf 1999; Murthy et al. 2000). NPR-C receptors, which are disulfide-linked homodimers with a single transmembrane domain (Anand-Srivastava and Trachte 1993), contain a specific Gi-activator domain within the intracellular portion of the receptor, a motif that was first described for the insulin-like growth factor receptor (Okamoto et al. 1990). In NPR-C, it is a 17 amino acid sequence (R469–R485) within the 37 amino acid intracellular domain that is responsible for the activation of Gi (Pagano and Anand-Srivastava 2001; Zhou and Murthy 2003). In this way, natriuretic peptides, when bound to NPR-C, are able to activate Gi proteins and decrease cAMP levels via the inhibition of adenylyl cyclase (Anand-Srivastava et al. 1996; Pagano and Anand-Srivastava 2001). We have recently shown that CNP has a potent and selective inhibitory effect on L-type calcium channels (ICa(L)) in the heart. This effect is mediated by this NPR-C/Gi signaling pathway (Rose et al. 2003, 2004b).
MNCs are potently affected by a number of neuropeptides that can have pronounced effects on hormone release. Furthermore, several peptide signals that modulate the release of VP and oxytocin (OT) have been described (Chakfe and Bourque 2000, 2001; Renaud and Bourque 1991). On this basis, and in accordance with what is known about NPR-C signaling in the heart, we hypothesized that CNP would have specific electrophysiological effects on MNCs that would require the activation of NPR-C. Our results show that CNP has a significant inhibitory effect on ICa(L) recorded in MNCs. Pharmacological experiments confirm that the CNP effect on ICa(L) is mediated by the NPR-C receptor and subsequent activation of a Gi protein. CNP, acting via the NPR-C receptor, can decrease the number of action potentials elicited during depolarizing stimuli in MNCs and also can significantly decrease the duration of the action potential. These results provide the first documentation of a functional role for the NPR-C receptor in the mammalian hypothalamus and demonstrate a novel ionic mechanism for this effect in the CNS. Some of these data have been presented in abstract form (Rose et al. 2004a).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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MNCs were isolated enzymatically from the supraoptic nucleus (SON) of the rat as described previously (Oliet and Bourque 1992). In summary, male Sprague-Dawley rats (75–125 g) were decapitated, and their brains were removed from the cranial vault. A coronal brain slice 2–3 mm in thickness was cut using a razor blade, and two blocks of tissue containing the SON were dissected out of this slice. These tissue blocks were incubated for 90 min at 34–35°C in 10 ml of oxygenated (100% O2) PIPES saline containing 7 mg of trypsin (Sigma type XI). PIPES saline consisted of (in mM) 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 20 1,4-piperazinediethanesulphonic acid (PIPES), and 25 glucose, pH was adjusted to 7. After enzyme treatment, the tissue blocks were rinsed twice with trypsin-free PIPES saline and triturated with fire polished pipettes (0.2–0.5 mm ID) to disperse the cells. The cell suspension was plated onto recording dishes for electrophysiological recordings.
Brain slice preparation
Coronal brain slices through the hypothalamus and containing the paraventricular nucleus (PVN) were prepared from male Sprague-Dawley rats (75–125 g) as described previously (Gordon and Bains 2003). Rats were anesthetized with pentobarbitol sodium (3 mg/kg) and decapitated, and the brains were excised into ice-cold high-sucrose slicing solution composed of (in mM): 87 NaCl, 2.5 KCl, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 1.25 NaH2PO4, 25 glucose, 75 sucrose, 3 pyruvic acid, and 1 ascorbic acid saturated with carbogen gas (95% O2-5% CO2). The brain was blocked and mounted on a vibrating slicer (Leica) with the tissue submerged in slicing solution for the duration of the slicing procedure. The hypothalamus was sliced in the coronal plane, and each slice (300 µM in thickness) was further hemisected and incubated in artificial cerebrospinal fluid (ACSF) composed of (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 10 glucose, and 1 ascorbic acid, saturated with carbogen gas and maintained at 32.5°C. Hypothalamic slices were incubated for 60 min before any electrophysiological recordings were made. All tissue preparations and animal handling procedures were in accordance with the guidelines set out by the University of Calgary animal care facility.
Electrophysiology
Enzymatically isolated MNC somata containing one to three short processes could be easily identified based on size and shape when viewed using an inverted microscope (Nikon Diaphot). Previous studies have established that >96% of enzymatically isolated cells from the SON having a cross-sectional area >160 µm2 are MNCs (Oliet and Bourque 1992). To be certain we were recording from isolated MNCs, only cells with a cross-sectional area >200 µm2 and that possessed a characteristic oblong shape were used for electrophysiological experiments.
For in situ experiments, hypothalamic slices through the PVN were transferred to a recording chamber mounted on an upright microscope (AxioscopeII FS Plus, Zeiss) with infrared differential interference contrast. MNCs were identified visually based on location in the PVN and individual cell morphology as described above. MNCs could also be identified by their prominent delay to first spike in response to depolarizing current pulses (Tasker and Dudek 1991). All electrophysiological recordings were made at 32.5°C.
The whole cell configuration of the patch-clamp technique (Hamill et al. 1981) was used for current- and voltage-clamp recordings from the somata of MNCs. Micropipettes for electrophysiological recordings were pulled from borosilicate glass (with filament, OD 1.5 mm, ID 0.86 mm, Sutter Instrument Co.) using a Flaming/Brown pipette puller (model P-89, Sutter Instrument Co.). The resistance of these pipettes was between 3 and 8 M
when filled with recording solution. The pipette solution for recording action potentials consisted of the following (in mM): 116 potassium gluconate, 2 MgCl2, 8 NaCl, 1 EGTA, 4 ATP (potassium salt), 0.3 GTP (sodium salt), and 10 HEPES. Hypothalamic slices were superfused with ACSF for recording action potentials.
For measurement of ICa(L) and ICa(T), the external bathing solution consisted of (in mM) 126 NaCl, 2.5 TEA-Cl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose. This solution was bubbled with carbogen gas to equilibrate pH. The pipette filling solution for measuring Ca2+ currents was composed of (in mM) 116 CH3CsO3S, 4 NaCl, 2 MgCl2, 1 EGTA, 4 ATP (sodium salt), 0.3 GTP (sodium salt), and 10 HEPES.
Recording pipettes were positioned using a micromanipulator (Burleigh) mounted on the microscope stage. Seal resistances were in the range of 2–10 G. Rupturing the cell membrane in the patch resulted in access resistances of 5–15 M. Series resistance compensation was 80–85% using an Axopatch 200B amplifier (Axon Instruments). Cell capacitance was 20–30 pF for enzymatically isolated MNCs and 40–80 pF for MNCs in situ. Current- and voltage-clamp signals were digitized using a Digidata 1322A interfaced with pCLAMP 8 software (Axon Instruments) and stored on computer for analysis off-line.
Action potentials were recorded by applying 500-ms depolarizing current pulses in 10-pA increments. Using this protocol, trains of action potentials were recorded a minimum of three times in control conditions and after application of CNP or cANF at 1, 3, and 5 min of drug application to ensure the responses were comparable. Only MNCs that had a stable resting membrane potential less than –50 mV were used. Peak ICa(L) was recorded by first applying a depolarizing voltage clamp step from –70 to –40 mV for 250 ms to inactivate T-type calcium current (Ertel et al. 1997). Immediately after this prepulse, 250-ms voltage-clamp steps were applied from –60 to +70 mV in 10-mV increments. The peak inward current and current-voltage (I-V) relations were plotted. All measurements of ICa(L) and ICa(T) were made in the presence of 1 x 10–6 M TTX to ensure blockade of voltage-gated sodium currents.
Drugs and chemicals
All chemicals and drugs were purchased from Sigma Chemical (St. Louis, MO) with the exception of TTX obtained from Alomone Labs (Jerusalem, Israel) and CNP and the NPR-C agonist, cANF purchased from Peninsula Laboratories (San Carlos, CA). The Gi-activator peptide, provided by M. B. Anand-Srivastava (Pagano and Anand-Srivastava 2001), was synthesized by standard solid-phase techniques and purified (95–99%) by high-performance liquid chromatography. The Gi-activator peptide was stored in solution at –70°C.
Statistical analysis
Summary data are presented as means ± SE. The data were analyzed using an ANOVA with Dunnett’s multiple comparison procedure (in most cases) or a paired Student’s t-test (Fig. 8; Table 1) to identify significant differences. In all cases, a P value <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Initial experiments sought to confirm previous work that identified specific voltage-gated calcium currents in the somata of MNCs (Fisher and Bourque 1995, 1996). ICa(T) is identified by its low threshold of activation and rapid inactivation kinetics, whereas ICa(L) is identified based on a high threshold of activation and much slower inactivation kinetics (Catterall 2000). Our experiments confirmed these findings in isolated MNCs and in MNCs in an in situ slice preparation. Sample recordings of calcium currents and their respective time-courses of inactivation (
; measured from the exponential curves shown in red) are presented for isolated MNCs (Fig. 1A) and in situ MNCs (Fig. 1B). In the isolated neuron in Fig. 1A, ICa(T) is observed during a voltage-clamp step to –40 mV ( = 8.1 ms), and ICa(L) is observed during a subsequent voltage-clamp step to 0 mV ( = 93.3 ms). Figure 1B shows similar voltage-clamp steps (note slightly different time scale) for an MNC in situ. In this neuron, ICa(T) has a of inactivation of 15.2 ms, and ICa(L) has a of inactivation of 81.8 ms. Summary data for of inactivation are presented in Fig. 1C. On average, ICa(T), measured during a voltage-clamp step to –40 mV, had a of inactivation of 25.2 ± 2.9 ms (n = 25 MNCs). of inactivation for ICa(L), measured during a voltage-clamp step to 0 mV, was 131.2 ± 10.9 ms (n = 29 MNCs). There was no significant difference between isolated and in situ MNCs, thus the data were combined.
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). Figure 1D shows sample ICa(L) recordings, as well as averaged time-course data for peak ICa(L), measured in the absence of any pharmacological agents to ensure our recording conditions do not result in significant rundown of the current. The representative traces are from a single MNC at the start of the experiment (trace 1) and after 300 s of recording (trace 2). In this neuron peak, ICa(L) was approximately –14 pA/pF in both recordings. On average, ICa(L) was stable with current densities of –15 to –20 pA/pF over 300 s (n = 6 MNCs). These recordings (Fig. 1D) showed that ICa(L) can be recorded in isolated MNCs for >5 min without a significant change in current magnitude. Figure 1E shows sample measurements of ICa(L) in control conditions (left) and after the application of nicardipine (right). The time-course, also shown in Fig. 1E, shows that nicardipine decreased ICa(L) from approximately –28 to –12 pA/pF in this MNC. These data are representative of nicardipine measurements made on five MNCs. On average, nicardipine, at a concentration of 1 x 10–5 M, decreased ICa(L) by 53.3 ± 8.4% (P < 0.05, n = 5 MNCs, Fig. 3B).
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To explore the electrophysiological effects of CNP on MNCs, voltage-clamp measurements of ICa(L) were made on enzymatically isolated MNCs. Figure 2, A and B, shows the effect of CNP (2x 10–8 M) on peak ICa(L) measured during a 250-ms voltage-clamp step to 0 mV, following a prepulse to –40 mV for 250 ms. This CNP dose is near the EC50 value for CNP effects evaluated in biochemical studies of natriuretic peptide signaling (Anand-Srivastava et al. 1990, 1996) and also within the range of natriuretic peptide doses shown to inhibit hormone release from MNCs in the hypothalamus (Yamamoto et al. 1991, 1997). Therefore this CNP dose is used throughout this study. Sample recordings (Fig. 2A) show that CNP decreased peak ICa(L) from –20 to –12 pA/pF in this MNC. The summary I-V curve (Fig. 2B) shows that, on average, CNP inhibited peak ICa(L) from –27.0 ± 8.0 to –10.1 ± 4.0 pA/pF (n = 5 neurons; P < 0.05).
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, 2004b). The next series of experiments evaluated this possibility in isolated MNCs. Figure 2, C and D, shows the effects of cANF (2 x 10–8 M) on ICa(L) in isolated MNCs. cANF is a ring-deleted analogue of atrial natriuretic peptide that has no ability to alter cGMP signaling through the binding of NPR-A or NPR-B (Anand-Srivastava et al. 1990). Rather, cANF has been shown to inhibit the cAMP signaling cascade when it binds to NPR-C (Anand-Srivastava et al. 1996; Pagano and Anand-Srivastava 2001; Zhou and Murthy 2003). Data from a representative MNC are shown in Fig. 2C. In this neuron, the NPR-C agonist cANF decreased peak ICa(L) from –22 to –12 pA/pF. The summary I-V curve (Fig. 2D) shows that, on average, cANF decreased peak ICa(L) from –27.7 ± 7.7 to –12.4 ± 3.6 pA/pF (n = 6 neurons; P < 0.05). ICa(L) was stable at this reduced amplitude for >30 min of washout. Partial recovery of ICa(L) current magnitude could be observed when the peptide was washed for >30 min (data not shown).
Effect of CNP is occluded by nicardipine
To determine if the NPR-C–mediated effect of CNP was specific to ICa(L), we performed an occlusion experiment in which nicardipine (1 x 10–5 M) was applied to MNCs before cANF (2 x 10–8 M) superfusion. In the representative traces and time-course experiment shown in Fig. 3A, nicardipine decreased peak ICa(L) from –20 to –10 pA/pF over the course of 350 s. Subsequent superfusion of this neuron with cANF (in combination with nicardipine) for another 350 s failed to elicit additional effects on peak current. On average, nicardipine inhibited peak ICa(L) by 53.8 ± 8.4%. Inhibition of peak ICa(L) in the presence of cANF and nicardipine was 58.8 ± 7.3%. There was no significant difference between the effects of nicardipine alone and the effects of nicardipine in combination with cANF (P > 0.05, n = 5 MNCs, Fig. 3B). These data show that the effect of cANF is occluded by pretreatment with nicardipine and suggest that the NPR-C–mediated effect of CNP is selective for ICa(L).
Effects of CNP on ICa(L) in MNCs in situ
We next examined the effects of CNP on MNCs in an acutely prepared hypothalamic slice preparation. Voltage-clamp measurements of the effect of CNP on ICa(L) in MNCs in situ are presented in Fig. 4. Representative raw data are shown in Fig. 4A; this data corresponds to the time-points indicated on the time-course experiment shown in Fig. 4B. In this MNC, ICa(L) was reduced from –20 to –4 pA/pF over the course of 300 s after the application of CNP (2 x 10–8 M). The summary I-V curve, which is shown in Fig. 3C, shows that on average CNP reduced peak ICa(L) in MNCs from –27.4 ± 2.3 to –15.6 ± 2.3 pA/pF (n = 8 neurons; P < 0.05).
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50% (compare Fig. 2 to Figs. 4 and 5). These electrophysiological findings (Figs. 2, 4, and 5) suggest that CNP specifically inhibits ICa(L) in MNCs by activating the NPR-C receptor. Although two populations of MNCs exist (those that secrete VP and those that secrete OT) we could not distinguish between these two phenotypes based on their responses to CNP. A careful analysis of every MNC treated with CNP or cANF (isolated and in situ) revealed that the inhibition of ICa(L) in each cell fell with a range of about 40–70% (n = 29 MNCs; data not shown). There was no obvious separation of responses into more than one group; therefore it is likely that ICa(L) is similarly inhibited in MNCs that secrete VP or OT. CNP does not affect ICa(T)
As shown in Fig. 1, and by others (Fisher and Bourque 1995, 1996), the somata of MNCs also express T-type Ca2+ channels. The effects of the NPR-C agonist, cANF (2 x 10–8 M), on ICa(T), which was measured during a voltage-clamp step to –40 mV for 250 ms in the presence of TTX (1 x 10–6 M), are shown in Fig. 6. In this neuron peak ICa(T) was not affected by application of cANF. The current was stable throughout the duration of the 500 s experiment. This result suggests the effects of CNP are specific to ICa(L) without altering another voltage-sensitive Ca2+ channel, ICa(T). Data are representative of measurements made on nine MNCs in situ (refer also to Fig. 8).
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Although it has been traditionally denoted as a "clearance receptor" (Levin et al. 1998), NPR-C is now known to contain a specific Gi-activator sequence. Specifically, the intracellular portion of NPR-C contains a 17 amino acid domain that is both necessary and sufficient for activation of Gi proteins in the heart (Pagano and Anand-Srivastava 2001; Zhou and Murthy 2003).
To provide further evidence for an NPR-C–mediated effect on ICa(L) in MNCs a synthetic Gi-activator peptide (consisting of the same 17 amino acid sequence shown to activate Gi in the heart) was included in the pipette solution. The Gi-activator peptide (1x 10–7 M) was able to enter MNCs under conventional whole cell recording conditions. Raw data and time-course effects of the Gi-activator peptide are shown in Fig. 7 A. Over the course of 300 s, the Gi-activator peptide decreased ICa(L) from approximately –23 to –5 pA/pF. This result is representative of measurements made on eight MNCs in which the average inhibition of peak ICa(L) was 68.0 ± 3.2% (P < 0.05, Fig. 8). Previous studies using sensitive biochemical assays have established that scrambled controls of this Gi-activator peptide have no effect on Gi protein/adenylyl cyclase activity (Pagano and Anand-Srivastava 2001).
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; Pagano and Anand-Srivastava 2001) and alter cAMP signaling. Accordingly, we hypothesized that the inhibition of ICa(L) by the Gi-activator peptide should be antagonized in the presence of elevated cAMP levels. To test this possibility, both the Gi-activator peptide (1 x 10–7 M) and cAMP (1 x 10–5 M) were added to the recording pipette with the expectation that each compound would enter the cell under whole cell recording conditions. Representative raw data and the time-course experiment are shown in Fig. 7B. At the onset of recording peak ICa(L) was approximately –18 pA/pF. After 400 s of exposure to the Gi-activator peptide and cAMP peak ICa(L) was –15 pA/pF. These data are representative of measurements made on six neurons in which, on average, a nonsignificant (P > 0.05) reduction in peak ICa(L) of 9.1 ± 2.1% was observed in the presence of the Gi-activator peptide and cAMP. Therefore cAMP blocked the action of the Gi-activator peptide on ICa(L) (refer to Fig. 7A). A summary of all experiments on ICa(L) and ICa(T) is presented in Fig. 8. CNP, applied in low nanomolar concentrations, significantly and selectively decreases ICa(L). This effect is mediated by the NPR-C receptor as evidenced by the experiments with cANF and the Gi-activator peptide. A nonsignificant (P > 0.05) 10% reduction in ICa(L) was observed when the Gi-activator peptide (1 x 10–7 M) was applied in conjunction with cAMP (1 x 10–5 M). Similarly, a nonsignificant (P > 0.05) 10–15% reduction in ICa(T) was observed in all experimental measurements of this calcium current. Because it occurred in all treatment groups, we suggest that this 10–15% reduction is likely a small run-down of the current over time rather than a specific effect of CNP.
Effect of CNP on action potential firing and action potential duration
Because ICa(L) is an important determinant of action potential (AP) firing (Bourque and Renaud 1985b; Fisher and Bourque 2001), experiments were undertaken to determine if the NPR-C receptor affects AP firing in MNCs in situ. Figure 9A shows the ability of cANF (2 x 10–8 M) to significantly inhibit the number of APs observed during a 60-pA depolarizing stimulus lasting 500 ms. cANF caused a 20% reduction in the number of APs observed in this neuron (from 53 APs in control conditions to 44 after superfusion with cANF). There was no significant difference between the effects of CNP and the NPR-C agonist, cANF, on AP firing. On average, the number of APs elicited during a 500 ms depolarizing stimulus was decreased in the presence of CNP and cANF by 25 ± 5% (n = 4 MNCs for CNP and 5 MNCs for cANF; P < 0.05).
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; Bourque and Renaud 1990). As such, we wanted to test the hypothesis that the inhibition of ICa(L) by CNP and cANF would result in an inhibition of APD50 in MNCs. The effect of the NPR-C agonist, cANF (2 x 10–8 M), on APD50 is presented in Fig. 9C. The superimposed action potentials show that the APD50 values in the presence of cANF are decreased compared with control conditions. Summary data for the effects of CNP and cANF on APD50 in MNCs are presented in Table 1, which shows that APD50 is significantly decreased in all action potentials that were measured in the presence of CNP or cANF compared with controls. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Magnocellular neurosecretory cells express a number of different voltage-sensitive Ca2+ channels including L-, T-, N-, and P-type Ca2+ channels. These Ca2+ channel subtypes are distributed in a heterogeneous way across the anatomical regions of the neuron. While the N- and P-type Ca2+ channels are located mainly in presynaptic nerve terminals, the L- and T-type Ca2+ channels are localized to the soma of MNC neurons (Fisher and Bourque 1995, 1996). Our data confirm that ICa(L) and ICa(T) can be identified in the somata of MNCs based on their respective time-courses of inactivation and sensitivity to specific pharmacological blockers.
Based on our papers showing CNP inhibits ICa(L) in the heart (Rose et al. 2003, 2004b), we hypothesized that this Ca2+ current would be similarly modulated in the hypothalamus. The data in Figs. 2, 4, and 5 show that ICa(L) in MNCs is inhibited by 50% after superfusion with CNP (2 x 10–8 M). Superfusing MNCs with cANF resulted in a similar pattern of inhibition of ICa(L), thus indicating that NPR-C is a physiologically active receptor in the mammalian hypothalamus. The NPR-C–mediated effects of CNP appear to be specific to ICa(L), because another time- and voltage-sensitive Ca2+ channel in the soma of MNCs, ICa(T), was not altered by superfusion with cANF (Fig. 6). In addition, although other high-voltage activated Ca2+ channels have been identified in MNC somata (Fisher and Bourque 1995, 1996), the experiment presented in Fig. 3, in which the effect of cANF on ICa(L) was occluded by nicardipine, indicates that the NPR-C–mediated effect of CNP is selective for ICa(L). It is not known whether CNP has a presynaptic effect on voltage-sensitive Ca2+ channels in MNC nerve terminals.
Another possible interpretation of our results is that CNP is, in fact, blocking much more than 50% of ICa(L). Other high-voltage activated Ca2+ currents are expressed in MNCs (Fisher and Bourque 1995, 1996). Therefore it may be that nicardipine blocks the majority of ICa(L) and that the current remaining during our voltage-clamp protocol is a different high-voltage activated Ca2+ conductance. In this case, because the effects of CNP are completely occluded by nicardipine (Fig. 3), CNP would be inhibiting ICa(L) by much more than 50% at the dose used in this study.
In the heart the NPR-C receptor is known to inhibit adenylyl cyclase via the activation of a pertussis toxin–sensitive Gi protein (Anand-Srivastava and Cantin 1986; Anand-Srivastava et al. 1987, 1996). This occurs through a specific Gi-activator domain (R469–R485) within the 37 amino acid intracellular portion of the NPR-C receptor. This Gi-activator sequence is characterized by the presence of two NH2-terminal basic residues and a COOH-terminal BBXXB motif, where B and X are basic and nonbasic residues, respectively (Pagano and Anand-Srivastava 2001; Zhou and Murthy 2003). In this way, when natriuretic peptides bind to NPR-C, Gi proteins are activated, and cAMP levels decrease. Applying the Gi-activator peptide to MNCs via the recording pipette (Fig. 7) resulted in a significant reduction of ICa(L), confirming the role of Gi in the NPR-C–mediated effect of CNP on ICa(L).
The ability of CNP and NPR-C to alter adenylyl cyclase and cAMP levels may explain the specificity of the effects of CNP on calcium currents. ICa(L) is known to be sensitive to changes in cAMP. Specifically, increasing or decreasing cAMP will alter the level of phosphorylation of ICa(L) by protein kinase A, thereby altering the open probability of the channel and increasing or decreasing current magnitude (Catterall 2000). Conversely, ICa(T) is insensitive to changes in intracellular cAMP (Ertel et al. 1997).
Effects of CNP on MNC excitability
We have shown that CNP and the NPR-C agonist, cANF, significantly inhibit action potential firing and APD50 in MNCs in an in situ hypothalamic slice preparation. It is important to note that cANF has no capacity to activate the NPR-B-cGMP pathway. Instead, it strongly inhibits adenylyl cyclase after binding to NPR-C (Anand-Srivastava et al. 1990), indicating the effect of CNP is mediated by the NPR-C receptor.
The electrical activity of MNCs changes in response to altered physiological conditions. Furthermore, the pattern of electrical activity has a direct influence on the pattern of hormone release from MNC terminals in the neurohypophysis (Poulain and Wakerley 1982; Renaud and Bourque 1991). The amount of VP and OT secreted is modulated by the frequency and duration of action potentials and the pattern of electrical activity in MNC nerve terminals. VP release, for example, is augmented when a pattern of phasic bursting is observed (Bourque and Renaud 1990; Poulain and Wakerley 1982; Renaud and Bourque 1991).
MNCs exhibit a significant broadening of successive action potentials in a train due to the activation of voltage-gated calcium channels (Bains and Ferguson 1999; Bourque and Renaud 1985a). This phenomenon is thought to be an important factor contributing to the frequency and pattern-dependent potentiation of hormone release in the nerve terminals (Fisher and Bourque 2001; Renaud and Bourque 1991). Based on these data, we suggest that the NPR-C receptor can mediate a CNP-induced decrease in action potential frequency and duration (refer to Fig. 9). This change in excitability and APD50 could account for the ability of this neuropeptide to inhibit hormone release from MNCs. Both ANP and BNP have been implicated in having a similar inhibitory effect on MNCs (Standaert et al. 1987; Yamamoto et al. 1991). Since NPR-C binds all of these natriuretic peptides with equal affinity (Levin et al. 1998), it may be that this receptor mediates changes in action potential firing by any natriuretic peptide.
In addition to being supported by calcium currents (Bourque and Renaud 1985b), action potential firing in neurons, including MNCs, is also modulated by specific calcium-sensitive potassium channels (Hatton and Li 1998; Stocker 2004). In MNCs, large and small conductance calcium-sensitive potassium channels, called BK and SK channels, respectively, have been shown to regulate repolarization of action potentials and generation of afterhyperpolarizations following trains of spikes. Modulation of BK and SK channels influences spike frequency accommodation and phasic or bursting firing patterns in MNCs (Hatton and Li 1998). It is not known whether the CNP and cANF effects on action potential firing observed in this study (Fig. 9) are due directly to an inhibition of ICa(L), or due to indirect effects of a change in ICa(L) on BK and/or SK channels, or a combination of both.
Our data indicate that CNP inhibits ICa(L) without modulating other voltage-gated Ca2+ currents. However, it is possible that CNP modulates other ion currents in MNCs, which could potentially contribute to the effects on action potential firing and duration presented in Fig. 9. For example, MNCs express a nonselective cation conductance that is known to be modulated by several neuropeptides, including angiotensin and neurotensin (Chakfe and Bourque 2000, 2001). We have not studied the ability of CNP to modulate these other ion currents in this study.
Significance of CNP effects on MNCs
CNP levels, as well as those of the other natriuretic peptides, are significantly elevated during congestive heart failure (Richards 2004; Wei et al. 1993). Because of this, plasma BNP levels are now used as a clinical marker and predictor of the onset of this disease (Richards 2004). VP synthesis, which is also significantly increased in heart failure, has been implicated in the pathophysiology of this syndrome (Lee et al. 2003). Binding of VP to the V1a and V2 receptor subtypes seems to modulate several processes thought to play a role in the progression of the disease; therefore VP antagonists are currently under development as potential therapeutic agents for the treatment of heart failure (Lee et al. 2003). It is possible that the increased production of natriuretic peptides during heart failure may serve to inhibit VP secretion and delay the progression of the disease. Our identification of the role of NPR-C in mediating CNP effects on MNCs, and potentially VP release, may provide new possibilities for pharmacological intervention in heart failure patients.
In summary, our results describe a novel role for the NPR-C receptor in MNCs of the hypothalamus. Specifically, ICa(L) is strongly and selectively inhibited by CNP via an NPR-C/Gi protein signaling mechanism. As a consequence, CNP can inhibit the excitability of MNCs and shorten the duration of the action potential. These findings show a functional role for the NPR-C receptor in the hypothalamus and provide new insights into an essential aspect of mammalian hypothalamic function.
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Address for reprint requests and other correspondence: J. S. Bains, Dept. of Physiology and Biophysics, Faculty of Medicine, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada (E-mail: jsbains{at}ucalgary.ca)
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