Inhibition of Calcium Currents in Rat Colon Sensory Neurons by K- But Not µ- or delta -Opioids

X. Su1, R. E. Wachtel2, 3, and G. F. Gebhart1

1 Department of Pharmacology and 2 Department of Anesthesia, College of Medicine, University of Iowa, Iowa City, 52242; and 3 Department of Veterans Affairs Medical Center, Iowa City, Iowa 52246

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Su, Xin, R. E. Wachtel, and G. F. Gebhart. Inhibition of calcium currents in rat colon sensory neurons by kappa - but not µ- or delta -opioids. J. Neurophysiol. 80: 3112-3119, 1998. We previously reported that kappa -, but not µ- or delta -opioid receptor agonists (ORAs) have selective, potentially useful peripheral analgesic effects in visceral pain. To evaluate one potential site and mechanism by which these effects are produced, we studied opioid effects on high-voltage activated (HVA) Ca2+ currents in identified (Di-I) pelvic nerve sensory neurons from the S1 dorsal root ganglion (DRG). Results were compared with opioid effects on cutaneous neurons from L5 or L6 DRG. Di-I-labeled DRG cells were voltage clamped (perforated whole cell patch clamp), and HVA Ca2+ currents were evoked by depolarizing 240-ms test pulses to +10 mV from a holding potential of -60 mV. Neither µ-ORAs (morphine, 10-6 M, n = 16; [D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin, 10-6 M, n = 12) nor delta -ORAs ([D-Pen2, D-Pen5] enkephalin, 10-7 M, n = 16; SNC-80, 10-7 M, n = 7) affected HVA Ca2+ currents in colon sensory neurons. In contrast, the kappa -ORAs U50,488 (10-6 M), bremazocine (10-6M), and nalBzoH (10-6 M) significantly attenuated HVA Ca2+ currents in colon sensory neurons; effects on cutaneous sensory neurons were variable. A nonreceptor selective concentration of naloxone (10-5 M) and nor-BNI (10-6 M), a selective kappa -opioid receptor antagonist, reversed the inhibitory effect of kappa -ORAs. In the presence of N-, P-, or Q-, but not L-type Ca2+ channel antagonists, the effect of U50,488 on HVA Ca2+ currents was significantly reduced. Pretreatment with pertussis toxin (PTX) prevented the inhibition by U50,488. These results suggest that kappa -opioid receptors are coupled to multiple HVA Ca2+ channels in colon sensory neurons by a PTX-sensitive G protein pathway. We conclude that inhibition of Ca2+ channel function likely contributes in part to the peripheral analgesic action of kappa -ORAs in visceral nociception.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

We recently documented that kappa -opioid receptor agonists (ORAs) selectively and dose-dependently attenuate pelvic nerve afferent fiber responses to noxious colorectal and urinary bladder distension (Sengupta et al. 1996; Su et al. 1997a,b). µ- and delta -ORAs exhibited no such peripheral actions, suggesting a potentially useful application for peripherally acting kappa -ORAs in the management of pelvic visceral pain. Neither the site(s) nor mechanism(s) of action of kappa -ORAs is presently known. One potential peripheral site of action addressed in this study is the dorsal root ganglion (DRG), which contains the cell bodies of pelvic nerve axons.

One effect of opioids is to reduce calcium-dependent neurotransmitter release from primary sensory neurons by reducing calcium entry through voltage-activated calcium channels (e.g., see North 1993). Recent studies revealed that both kappa - (Barro et al. 1995; Wiley et al. 1997) and µ-ORAs (Moises et al. 1994a,b; Nomura et al. 1994; Rusin and Moises 1995; Schroeder and McCleskey 1993; Schroeder et al. 1991; Wilding et al. 1995; Womack and McCleskey 1995) suppress whole cell calcium currents. In at least some DRG neurons, µ- and kappa -opioid receptors appear to be functionally coupled to a common pool of Ca2+ currents (Moises et al. 1994b).

The sensitivity of individual DRG neurons to modulation by ORAs varies widely, however. DRG neurons are a mixed population of cells, and the variable sensitivity is likely due to the variety in sensory modalities associated with the cells. There is little experimental data comparing the sensitivity of different DRG neurons to opioids based on their sources and organs of innervation. Most authors studied DRG cells at random, although Schroeder and McCleskey (1993) dissociated neurons according to the spinal lamina to which they likely projected and also injected Di-I into skeletal muscle to determine which neurons had innervated the muscle.

The purpose of this study was to examine opioid effects on voltage-activated Ca2+ currents in identified colon sensory neurons. We also compared the effects of opioids on colon sensory neurons with effects on a sample of cutaneous sensory neurons. Because previous reports documented that opioids have no effect on low-voltage activated (LVA) Ca2+ currents (Gross and MacDonald 1987; Moises et al. 1994b; Rusin and Moises 1995), this study focused on the effects of opioids on high-voltage activated (HVA) Ca2+ currents. We also investigated whether multiple Ca2+ currents were coupled to a kappa -opioid receptor and whether the inhibition by ORAs of Ca2+ currents occurs by means of a pertussis toxin (PTX)-sensitive guanine nucleotide-binding protein (G protein) pathway. A preliminary report of some of this work appeared in abstract form (Su et al. 1997c).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Labeling of colon or cutaneous sensory neurons

Male adult Sprague-Dawley rats weighing 250-300 g (Harlan, Indianapolis, IN) were anesthetized with sodium pentobarbital (Nembutal). For colon sensory DRG labeling, the descending colon was exposed under sterile conditions. Seventy microliters of the dicarbocyanine dye Di-I (1.1'-dioctadecyl-3,3,3,'3'-tetramethylindocarbocyanine methanesulfonate; 25 mg in 0.5 ml methanol) was injected at multiple sites into the smooth muscle of the colon through a 30G needle. The surgical incision was closed, and rats were allowed to recover for 1-2 wk to permit Di-I to be transported to the cell soma (e.g., S1 DRG). For cutaneous sensory DRG labeling experiments, Di-I was injected bilaterally into the skin of the outer and inner thigh. The care and use of rats conformed to the standards established by the U.S. Department of Agriculture and by the National Institutes of Health. All protocols were approved by the University of Iowa Institutional Animal Care and Use Committee.

Cell dissociation and culture

Rats were anesthetized with sodium pentobarbital (Nembutal), and the S1 (for colon sensory neurons) or the L5 and L6 (for cutaneous sensory neurons) DRGs were removed bilaterally, stripped of their connective tissue capsules, transferred into ice-cold culture media, and minced with microscissors. The ganglia tissue was digested in modified L-15 culture media containing collagenase (type Ia, 1 mg/ml), trypsin (type III, 1 mg/ml), and deoxyribonuclease (type IV, 0.1 mg/ml) at 37°C for 50 min. The chemical digestion was terminated by adding soybean trypsin inhibitor (2 mg/ml) and bovine serum albumin (1 mg/ml). The tissue fragments were then gently triturated with a siliconized sterile Pasteur pipette and centrifuged at 800 g for 5 min. The neurons were resuspended in modified L-15 media supplemented with 5% rat serum and 2% chick embryo extract and plated onto poly-L-lysine-coated glass coverslips. Neurons in the culture media were kept at 37°C in an incubator under a 95% air-5% CO2 atmosphere saturated with water vapor. Cells were studied within 24 h. Only Di-I-labeled DRG cells, identified by their red-orange color under Hoffman contrast optics (×400) in fluorescent light with a Rhodamine filter (excitation wavelength ~546 nM and barrier filter at 580 nM), were selected for study.

Whole cell current recordings

Neurons were studied by using the nystatin perforated whole cell patch-clamp technique. Coverslips containing cells were transferred into a 1-ml recording chamber with medium of the following composition (in mM): 110 tetraethylammonium, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10 D-glucose, and 5 BaCl2; the pH was adjusted to 7.35 with CsOH, and the osmolarity was adjusted to 320 mosm/l with sucrose. The tips of patch electrodes were filled with a solution containing (mM): 120 CsCl, 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 HEPES; the pH was adjusted to 7.25, and the osmolarity was adjusted to 310 mosm/l. Electrodes were then backfilled with solution containing nystatin. A reference electrode was connected through a Ag-AgCl pellet to the bath solution via a CsCl agar bridge. The offset potential between the pipette and bath solution was zeroed before seal formation. Inward barium current through calcium channels will be referred to as calcium current.

Some dissociated neurons were pretreated with PTX (200 ng/ml) for 18-24 h before recording. PTX was also included in the bath solution and in the pipette solution (200 ng/ml) during recordings.

Recordings were made at room temperature. Currents were recorded with an Axopatch 1C patch-clamp, low-pass filtered at 5 kHz, digitized at 200 µs per point, and stored on a computer for later analysis. Pipette and whole cell capacitance and series resistance were corrected with compensation circuitry on the patch-clamp amplifier. In most cases, series resistance compensation of 88-92% was obtained without inducing significant noise or oscillation, resulting in a final series resistance averaging 4 MOmega . Pulse protocols and data collection were performed by pCLAMP software package (Axon Instruments). Calcium currents were corrected for leak and capacitative currents with a P/4 routine that involved subtraction of an appropriately scaled current elicited by a series of four small depolarizing prepulses, whose sum was equal in magnitude to the depolarizing test pulse.

For recording HVA Ca2+ currents, cells were held at a resting potential of -60 mV and depolarized by a series of 160-ms voltage-clamp command pulses to potentials ranging from -40 to +60 in 20-mV increments. The interval between pulses was 5 s. To study the time course of drug effects, voltage step commands of 240-ms duration were applied every 10 s from a resting potential of -60 mV to a test potential of +10 mV. For recording LVA Ca2+ currents, cells were held at a resting potential of -90 mV and depolarized by a series of 160-ms voltage-clamp command pulses to potentials ranging from -90 to -10 in 20-mV increments.

Data analysis

A neuron was considered sensitive to an ORA if the HVA Ca2+ current was inhibited to <90% of control. All group data are expressed as means ± SE. Results were analyzed with Student's t-test or an analysis of variance for repeated measures. A value of P < 0.05 was considered statistically significant.

Drugs

U50,488 [molecular weight (MW): 465.4, Research Biochemicals, (RBI), Natick, MA], bremazocine (MW: 351.9, RBI), and NalBzoH (MW: 445.52, RBI, dissolved in 2.5% acetic acid to make a 10-3 M stock solution) were selected for study as representative of putative kappa 1-, kappa 2 - and kappa 3-ORAs (Clark et al. 1989; Gistrak et al. 1989; Paul et al. 1990; Price et al. 1989; Rothman et al. 1990; see Rothman 1994 for review). Other ORAs were morphine sulfate (µ-ORA; MW: 668.7, Merck Chemical Division, Merck, Rahway, NJ), DAMGO (µ-ORA; [D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin, MW: 523.7, RBI), DPDPE (delta -ORA; [D-Pen2, D-Pen5] enkephalin, MW: 645.8, RBI), and SNC-80 (delta -ORA; MW: 449.6, Tocris Cookson, St. Louis, MO, dissolved in 10% methanol). Antagonists were nor-BNI dihydrochloride (MW: 734.7, RBI) and naloxone hydrochloride (MW: 363.8, Sigma Chemical Co., St. Louis, MO). Nystatin (MW: 926.1, Sigma) stock solution contained 50 mg/ml in dimethyl sulfoxide and was diluted 1:300 in electrode solution immediately before use. PTX was obtained from Sigma.

Ca2+ channel antagonists were as follows (Rusin and Moises 1995; Wiley et al. 1997): omega -conotoxin MVIIA (MVIIA; N-type; MW: 2639.1; Sigma), omega -agatoxin IVA (Aga IVA; P-type; MW: 5202.3; CalBiochem-Novabiochem, La Jolla, CA), omega -conotoxin MVIIC (MVIIC; Q-type; MW: 2749.2; Sigma), and nifedipine (L-type; MW: 346.34; RBI; dissolved in 1% methanol before use).

All compounds were applied by addition to the bathing solution or by superfusion. In some experiments in which multiple Ca2+ channel antagonists were studied, short pulses of U50,488 (10-6 M; 1 s) were applied repeatedly by pressure ejection from a large-bore pipette placed near the cell.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Cell labeling

To estimate the proportion of S1 DRG cells that were labeled, we counted all cells found on eight coverslips. Of 3,552 neurons, only 1.2% (42) were labeled to indicate that they were colonic neurons. Colonic neurons (34 ± 0.3 µm, n = 391 total measured) were not different in size from unlabeled S1 DRG cells (29 ± 0.4 µm). When L5/L6 DRG neurons from the skin were compared with colonic neurons, there was no difference in mean cell diameter (31 ± 0.6 µm).

Properties of Ca2+ currents

Typical examples of both LVA and HVA Ca2+ currents are shown in Fig. 1. LVA Ca2+ currents were isolated by stepping from a holding potential of -90 mV to test potentials of -90 to -10 mV. Of 59 colon neurons sampled, 55 (93%) had classical LVA Ca2+ currents that inactivated rapidly (Fig. 1, left panels). Maximal current amplitude at -10 mV averaged -1714.6 ± 190 pA (range -103 to -5347 pA). The LVA Ca2+ current has also been called a T current because of its transient nature.


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FIG. 1. Two examples of Ca2+ currents in colon sensory dorsal root ganglion (DRG) cells. A: representative families of Ca2+ currents from 1 colon sensory neuron that has both low-voltage activated (LVA, left) and high-voltage activated (HVA, right) Ca2+ currents. B: representative family of Ca2+ currents from 1 colon sensory neuron that has only HVA calcium currents.

HVA Ca2+ currents were isolated by stepping from a holding potential of -60 mV to test potentials of -40 to +60 mV (Fig. 1, right panels). All colon sensory neurons had classical HVA Ca2+ currents which did not completely inactivate.

Effects of ORAs on HVA Ca2+ currents in colon DRG cells

Experiments were designed to determine effects of a variety of ORAs on HVA Ca2+ currents. Voltage steps of 240-ms duration were applied every 10 s from a resting potential of -60 mV to a test potential of +10 mV. At this holding potential, low-threshold Ca2+ currents were at least partially inactivated (Gross and MacDonald 1987; Moises et al. 1994b).

Figure 2A shows examples of data from a single cell that was serially exposed to the µ-ORA DAMGO (10-6 M), the delta -ORA SNC-80 (10-7 M), and the kappa -ORA bremazocine (10-6 M). Only bremazocine attenuated the HVA Ca2+ current, and its effect was partially reversible on washing. Data for a number of different agonists are summarized in Fig. 2B. The µ-ORAs morphine and DAMGO did not affect HVA Ca2+ currents at 10-6 M. The peptide delta -ORA DPDPE (10-7 M) and the nonpeptide delta -ORA SNC-80 (10-7 M) also did not have any effect on HVA Ca2+ currents. However, all kappa -ORAs tested (U50,488, bremazocine, and nalBzoH, all at 10-6 M) significantly inhibited peak HVA Ca2+ currents at all voltages tested (Fig. 2C).


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FIG. 2. A: representative data from a single colon sensory neuron exposed to DAMGO (µ), SNC-80 (delta ), and bremazocine (kappa ). Currents were elicited by stepping to a test potential of +10 mV. Only bremazocine attenuated HVA Ca2+ currents, and its effects were reversible on washing. B: summary of the effects of µ-ORAs (morphine and DAMGO), delta -ORAs (DPDPE and SNC-80), and kappa -ORAs (U50,488, bremazocine and nalBzoH) on HVA Ca2+ currents. Currents were inhibited only by kappa -ORAs. C: current-voltage (I-V) relationships for U50,488, bremazocine, and nalBzoH showing that the voltage dependence of channel activation was not altered. The number of cells is shown in each panel.

A total of 20 labeled neurons were each tested with all three types of ORAs (µ: morphine or DAMGO at 10-6 M; delta : DPDPE or SNC-80 at 10-7 M; and kappa : U50,488H, bremazocine, or nalBzoH at 10-6 M). HVA Ca2+ currents in 16 neurons (80%) were inhibited by kappa -ORAs, currents in 2 neurons (10%) were inhibited by both kappa - and delta - ORAs, and currents in 2 neurons (10%) did not respond to any of the ORAs tested.

The inhibition produced by kappa -ORAs was clearly dose dependent. Figure 3 illustrates results of an experiment in which several concentrations of bremazocine were superfused continuously into the recording chamber. Current amplitude decreased with increasing concentrations of bremazocine, and the inhibition was reversed on washing. Grouped data presented in Fig. 4 illustrate the concentration-dependent inhibition produced by all the kappa -ORAs tested and confirm the lack of effect in colon sensory neurons of the µ-ORA morphine and the delta -ORA SNC-80.


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FIG. 3. HVA Ca2+ currents recorded from a colon sensory neuron perfused with increasing concentrations of bremazocine. HVA Ca2+ currents were inhibited by bremazocine in a concentration-dependent manner. A: representative HVA Ca2+ currents sampled at the times corresponding to the arrows shown in B. B: time course of changes in HVA Ca2+ current amplitude as bremazocine concentration is increased. open circle : ~10 s.


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FIG. 4. The kappa -ORAs U50,488, bremazocine, and nalBzoH attenuated HVA Ca2+ currents in a concentration-dependent manner over the concentration range tested (10-8 to 10-5 M). Morphine and SNC-80 had no significant effects.

U50,488-mediated reduction of multiple Ca2+ currents

Multiple HVA Ca2+ currents were present in colon DRG cells, as evidenced by the partial block produced by several selective Ca2+ channels blockers. MVIIA (10 µM; N type), Aga IVA (200 nM; P type), MVIIC (1 µM; Q type), and nifedipine (10 µM; L type) were added sequentially in the recording chamber. Finally, cadmium was added to block toxin-resistant currents. In seven neurons, MVIIA (10 µM) irreversibly blocked 28.2 ± 6.9%, Aga IVA (200 nM) suppressed 24.6 ± 6.4%, MVIIC (1 µM) antagonized 16.8 ± 3.1%, and nifedipine (10 µM) blocked 13.5 ± 3.5% of the control whole cell Ca2+ currents. In addition, 17.6 ± 10.1% of the HVA Ca2+ currents was found to be cadmium sensitive but resistant to blockade by the four Ca2+ channel antagonists. Among the seven neurons, one did not express Q-type and one lacked L-type Ca2+ currents. In other experiments, nifedipine (10 µM) was added first, followed by MVIIC (1 µM); they antagonized 21.3 ± 4.6% (n = 6) and 20.4 ± 4.5% (n = 4) of the control whole cell Ca2+ currents, respectively.

To determine which components of the HVA Ca2+ currents were sensitive to U50,488, short pulses of U50,488 (10-6 M; 1 s) were applied repeatedly before and after selective blockade of N-, P-, Q-, and L-type currents (Fig. 5). Sequential application of N-, P-, and Q-type blockers all reduced the inhibition produced by U50,488, whereas application of an L-type blocker had no effect on the inhibition. Figure 5C shows summary data in which nifedipine, an L-type blocker, was applied before the other antagonists, confirming that it did not reduce the response to U50,488. The U50,488-sensitive HVA Ca2+ current contains N-, P-, and, Q-type calcium channels but not L-type channels.


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FIG. 5. Effects of U50,488 on HVA Ca2+ currents after addition of Ca2+ channel antagonists to the recording chamber. A: representative cell illustrating the time course of Ca2+ current amplitude on sequential application of N-type (omega -conotoxin MVIIA), P-type (omega -agatoxin IVA; Aga IVA), Q-type (omega -conotoxin MVIIC), and L- type (nifedipine) blockers. Short pulses of U50,488 (10-6 M; 1 s) were applied at the times indicated by each filled square. U50,488 inhibited HVA Ca2+ currents by 488 pA in control. After administration of N-, P-, and Q-type Ca2+ channel antagonists, the inhibitions produced by U50,488 were 364, 124, and 50 pA, respectively. B and C: summary data demonstrating that the inhibition produced by U50,488 was reduced significantly after selective blockade of N-, P-, or Q- but not L-type channels. * P < 0.05. In B, blockers were applied in the order N, P, Q, and L. In C, an L-type blocker was applied first, followed by a Q-type blocker.

Involvement of PTX-sensitive G protein

Cells were pretreated with PTX for ~20 h at 37°C. PTX was also added to pipette and bath solutions. HVA Ca2+ currents in 11 neurons tested were not different from those in control neurons. However, the inhibition produced by U50,488 (10-6 M) was markedly attenuated after treatment of neurons with PTX, averaging 94.0 ± 2.7% of control (P < 0.05) compared with 75.2 ± 3.0% of control without PTX treatment (n = 43), suggesting that the U50,488-induced decrease in HVA Ca2+ currents may be mediated via activation of a PTX-sensitive G protein.

Effects of opioid receptor antagonists

Additional experiments tested the specificity of the effect of kappa -ORAs by determining their sensitivity to blockade by opioid receptor antagonists. Figure 6 shows HVA Ca2+ currents from a typical neuron. The current was inhibited by bremazocine (10-6 M) but partially recovered on washing. Subsequent application of nor-BNI (10-6 M), a kappa -opioid receptor antagonist, had no direct effect on the HVA Ca2+ current but blocked the inhibitory effects of bremazocine. Summary data in Fig. 7A demonstrate that pretreatment with nor-BNI (10-6 M) antagonized the inhibitory effects of all three kappa -ORAs tested. In addition, a concentration of naloxone (10-5 M) that is opioid receptor nonselective also reduced the attenuation produced by all three of the kappa -ORAs tested (Fig. 7B). A µ-receptor selective concentration of naloxone (10-8 M), however, did not block the effect of U50,488 (Fig. 7C).


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FIG. 6. Example of a colon sensory neuron showing attenuation of HVA Ca2+ currents by 10-6 M bremazocine (kappa ). Effects were reversible on washing. Nor-BNI (10-6 M) alone had no effect on HVA Ca2+ currents but prevented the inhibitory effect of bremazocine.


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FIG. 7. Comparison of the effects of kappa -ORAs before and after treatment with receptor antagonists. In each panel, the unfilled bar represents HVA Ca2+ current amplitude, as a percent of control, in the presence of a kappa -ORA. The filled bar represents current amplitude in the presence of the same kappa -ORA but after cells were pretreated with an antagonist. A: nor-BNI (10-6 M), a selective kappa -opioid receptor antagonist, prevented the inhibitory effect of U50,488 in 4 of 6 cells tested, bremazocine in 5 of 7 cells tested, and nalBzoH in 5 of 5 cells tested. B: naloxone (10-5 M), at a nonselective opioid receptor antagonist concentration, reversed the inhibitory effect of U50,488 in 5 of 6 cells tested, bremazocine in 5 of 5 cells tested, and nalBzoH in 5 of 5 cells tested. C: naloxone (10-8 M), at a selective µ-opioid receptor antagonist concentration, did not block the inhibitory effects of U50,488 in 5 of 5 cells tested. * P <0.05 relative to agonist alone.

Effects of ORAs in cutaneous DRG cells

All the ORAs were also tested on HVA Ca2+ currents in labeled cutaneous neurons from L5 or L6 DRG. Results were variable. Morphine (10-6 M) slightly inhibited HVA Ca2+ currents in 5 of 10 cells tested, and the delta -ORAs DPDPE (10-7 M) and SNC-80 (10-7 M) inhibited currents in 4 of 19 cells tested. NalBzoH had inhibitory effects on HVA Ca2+ in four of nine cutaneous DRG cells, whereas other kappa -ORAs had no obvious effects.

Data for both colon and cutaneous DRG cells are summarized in Fig. 8 and Table 1.


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FIG. 8. Comparison of the effects of ORAs on HVA Ca2+ currents in colon (filled circles) and cutaneous (unfilled circles) DRG cells. Each symbol represents results from a single cell. The vertical boxes represent the median and the 25th and 75th percentiles. Dotted lines show the means. The error bars represent the 10th and 90th percentiles. Morphine and nalBzoH inhibited HVA Ca2+ currents in some cutaneous DRG cells. In general, only kappa -opioid receptors were found to be consistently coupled to HVA Ca2+ channels in colon sensory neurons. Data from PTX-treated DRG cells are also shown for comparison.

 
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TABLE 1. Effects of ORAs on HVA Ca2+ currents in colon and cutaneous sensory neurons

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The three kappa -ORAs but none of the µ- or delta -ORAs tested all produced similar concentration-related inhibition of HVA Ca2+ currents in colon sensory DRG neurons. In contrast, cutaneous DRG cells showed no consistent pattern of inhibition by any ORA. These results suggest specific roles for kappa  (or kappa -like) opioid receptors in the modulation of visceral nociception.

Opioid receptors

Several lines of evidence support the conclusion that inhibition of HVA Ca2+ currents by kappa -ORAs is mediated via activation of a kappa -opioid receptor. First, a high concentration of the opioid receptor antagonist naloxone (10-5 M) blocked the inhibition produced by U50,488, bremazocine, and nalBzoH, indicating that all the agonists were acting at opioid receptors. Second, a lower concentration of naloxone (10-8 M), corresponding to the reported equilibrium dissociation constant of this antagonist at µ-opioid receptors (see Williams and North 1984), was not able to block or reverse the inhibition produced by any of the kappa -ORAs. Administration of nor-BNI, which reversibly blocks kappa -opioid receptors (Spanagel et al. 1994; Takemori et al. 1988), abolished the effects of U50,488, bremazocine, and nalBzoH. Last, the delta - and µ-ORAs failed to inhibit HVA Ca2+ currents in most neurons, thus ruling out delta - and µ-receptors in the mediation of the inhibitory effects observed.

Comparison with pelvic nerve afferents

In our earlier studies, the kappa -ORAs U50,488H (kappa 1), U69,593 (kappa 1), U62,066 (kappa 1), bremazocine (kappa 2), and nalBzoH (kappa 3) dose-dependently attenuated pelvic nerve afferent fiber responses to colorectal distension, whereas neither µ-ORAs (morphine and fentanyl) nor delta -ORAs (DPDPE and SNC-80) affected afferent fiber responses to distension (Sengupta et al. 1996; Su et al. 1997b). The finding that kappa - but not µ- or delta -ORAs inhibit HVA Ca2+ currents in colon sensory DRG neurons suggests that inhibition of HVA Ca2+ currents may be a mechanism by which kappa -ORAs attenuate pelvic nerve afferent fiber responses. However, the mean maximum inhibition of pelvic nerve afferent fiber responses to colonic distension by U50,488, bremazocine, and nalBzoH was to 25-40% of control, whereas the mean maximum inhibition of HVA Ca2+ currents by the same kappa -ORAs (10-6 M) in this study was to 70-80% of control. Therefore the peripheral visceral antinociceptive effects of kappa -ORAs cannot be attributed solely to an action on HVA Ca2+ currents in the cell bodies of pelvic nerve neurons. Other mechanisms may also contribute to attenuation of pelvic nerve afferent fiber responses. Another difference between the results reported here and those of our in vivo experiments is that nor-BNI antagonized the effects of kappa -ORAs on HVA Ca2+ currents in the current experiments but was ineffective in blocking the effects of kappa -ORAs in the in vivo experiments (Sengupta et al. 1996; Su et al. 1997b). One possible explanation is that nor-BNI may not have had access to kappa -receptors in in vivo afferent fiber experiments. This seems unlikely because antagonists were given both acutely or as pretreatments (hours and days) in those experiments. Alternatively, it is possible that the kappa -receptor on DRG cells is different from the receptor studied in peripheral tissue (colon or bladder), although there is no evidence available to asses this possibility. It is also possible that the concentration of nor-BNI (10-6 M) used here may not be sufficiently selective for the kappa -opioid receptors present on the DRG cells and may have produced nonspecific block of the receptor.

Calcium currents

Multiple HVA Ca2+ currents were present in these cells, as evidenced by the partial block produced by several selective Ca2+ channel blockers. Currents consisted of 28% N-type, 25% P-type, 17% Q-type, 13% L-type, and 17% toxin resistant but cadmium sensitive. Although these proportions may have been slightly biased because blockers were always applied in the same order, the relative proportions of Ca2+ channel types are similar to those reported by other investigators with a more heterogeneous population of cells (Mintz et al. 1992; Rusin and Moises 1995; Wiley et al. 1997), except that we found a slightly lower proportion of N-type Ca2+ channels. The inhibition produced by U50,488 appears to be caused by a reduction in current through N-, P-, or Q- but not L-type channels.

Interactions between kappa -opioid receptors and voltage-dependent Ca2+ channels were reported previously (see North 1993 for overview). In mice, most kappa - and some µ- and delta -ORAs decrease the duration of DRG neuron somatic Ca2+-dependent action potentials, resulting in a decrease in Ca2+ entry and decreased neurotransmitter release (Werz and MacDonald 1985). MacDonald and Werz (1986) reported that dynorphin (kappa ) but not (leu)enkephalin (µ- and delta -) reduced voltage-dependent Ca2+ currents. In 12- to 14-day-old fetal rat DRG cells, kappa -opioid receptors are negatively coupled to the dihydropyridine class of voltage-dependent Ca2+ channels (i.e., L-type), which represents >90% of the channel population involved in Ca2+ entry on depolarization (Attali et al. 1989). In contrast, Wiley et al. (1997) reported that dynorphin modulates multiple Ca2+ currents, including N-, P- and Q- but not L-type channels in rat DRG. In other studies, L-type channels did not appear to be regulated by kappa -opioid receptors in DRG cells (Gross and MacDonald 1987; Gross et al. 1990). In a random sample of DRG neurons, Moises et al. (1994b) found that Ca2+ channels can be modulated by both µ- and kappa - but not delta -ORAs on the same sensory neurons. Thus depression of Ca2+ channel activity by ORAs probably involves multiple Ca2+ channels and multiple opioid receptor types.

Inhibitory coupling between kappa -opioid receptors and calcium channels involving a PTX-sensitive G-protein was documented in nodose neurons (Gross et al. 1990). Wiley et al. (1997) used intracellular dialysis with antibodies to determine that kappa -opioid receptors were coupled to the Go-subtype G protein in DRG cells. We found that treatment with PTX, which prevents activation of inhibitory Go/Gi-type G proteins, antagonized the inhibition of U50,488 on HVA Ca2+ currents in colon sensory neurons, suggesting that coupling of kappa -opioid receptors to multiple Ca2+ channels also involves PTX-sensitive G proteins.

Comparison with cutaneous sensory neurons

We also compared the effects of ORAs on HVA Ca2+ currents of colon sensory neurons with those of cutaneous sensory neurons. Morphine and nalBzoH both inhibited HVA Ca2+ currents in some neurons in the sample of labeled cutaneous neurons studied. In contrast to other reports (Schroeder and McCleskey 1993; Schroeder et al. 1991; Wilding et al. 1995; Womack and McCleskey 1995), we did not find a significant inhibition by the µ-ORA DAMGO. This may reflect the different cell culture conditions (e.g., we did not add nerve growth factor) or the DRG neurons recorded, which were typically chosen randomly from lumbar and thoracic regions.

In vivo studies on afferent fibers support our observations. For example, morphine (µ), DAMGO (µ), and U50,488 (kappa ) reduce spontaneous discharges of fine afferent fibers innervating the inflamed knee joint of the cat (Russell et al. 1987). Morphine (µ) and U69,593 (kappa ) but not DPDPE (delta ) suppress the spontaneous activity of saphenous nerve afferent fibers from injured tissue after irradiation of rat hind paw skin (Andreev et al. 1994). In the absence of tissue injury, however, opioids do not depress the spontaneous discharges of C-polymodal nociceptors in normal skin and do not depress their responses to noxious stimuli (Senami et al. 1986; Shakhanbeh and Lynn 1993). In the aggregate, previous and current results suggest that the receptor in the periphery at which kappa -ORAs act to produce significant attenuation of responses to noxious visceral distension is different from the cloned kappa -opioid receptor (see DISCUSSION in Su et al. 1997b). These results extend our earlier observations, suggesting that the principal site of action is peripheral to the DRG and that only a portion of the mechanism by which noxious visceroceptive input is modulated by kappa -ORAs is associated with the effects on HVA Ca2+ currents. The finding that intracolonic administration of kappa -ORAs also inhibits responses of peripheral nerve afferent fibers to colorectal distension suggests a peripheral site of action (Su et al. 1998).

    ACKNOWLEDGEMENTS

  The authors thank M. Burcham for production of the graphics, C. A. Whiteis for assistance in culturing cells, and Drs. Brett A. Adams and Kathleen A. Sluka for commenting on an earlier version of the manuscript.

  This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-19912.

    FOOTNOTES

  Address for reprint requests: X. Su, Dept. of Pharmacology, Bowen Science Building, The University of Iowa, Iowa City, IA 52242.

  Received 18 May 1998; accepted in final form 14 September 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society