Spinal Antinociceptive Action of an N-Type Voltage-dependent Calcium Channel Blocker and the Synergistic Interaction with Morphine

Experiments were conducted according to the protocol approved by the Sapporo Medical University Animal Care and Use Committee.

Male Sprague-Dawley rats (weighing 250–300 g, Japan SLC, Hamamatsu, Japan) were used. The animals were housed individually in a temperature-controlled (21+/-1 degree C) room with a 12-h light:dark cycle (lights on 7:00 AM-7:00 PM) and were allowed free access to food and water.

Using a modification of the method described by Bahar et al., [17]chronic lumbar intrathecal catheters were implanted in rats anesthetized with halothane. Briefly, a polyethylene catheter (PE-10, Clay Adams, Parsippany, NJ) was introduced 15 mm cephalad into the lumbar subarachnoid space at the L4-L5 vertebral level with the tip of the catheter located near the lumbar enlargement of the spinal cord. The distal end of the catheter was tunnelled subcutaneously to emerge at the neck. The volume of dead space of the intrathecal catheter was 10 micro liter. After the intrathecal catheters were implanted, the rats were housed in individual cages. At least 6 days of postsurgical recovery was allowed before animals were used in experiments. In the experiments, we used only animals that showed normal behavior and motor function and that had showed complete paralysis of tail and bilateral hind legs after administration of 2% lidocaine 10 micro liter through the intrathecal catheter. After the experimentation, in which the animals were used only once, each animal was killed by an overdose of halothane. The position of the catheter tip was verified at autopsy and the spread of injected material was determined by a postmortem intrathecal injection of 1% methylene blue (10 micro liter) followed by a flush of physiologic saline.

The tail flick (TF) and the mechanical paw pressure (MPP) tests were used to assess thermal and mechanical nociceptive thresholds, respectively. The TF test was employed by monitoring latency to withdrawal from the heat source (a 50-W projection lamp bulb) focused on the distal segment of the tail, using a thermal analgesimeter (KN-205E, Natsume, Tokyo, Japan). The location on the stimulated tail was systemically varied so that the same portion of the tail was not exposed repeatedly to the light source. The mean baseline TF latency in the experiment was 3.4 s (3.1–3.7 s) and a cutoff time of 10.0 s was adopted to minimize damage to the skin of the tail.

The MPP test was employed on the right hind paw of the animal, using a Randall Selitto type analgesimeter (TK-201, Unicom, Tokyo, Japan). The head and body of the rat was loosely wrapped in a soft cloth and gently held in the hand of the observer. The hind paw was then placed on a flat plastic surface and pressure was applied to the unrestrained dorsal surface of the hind paw by a small conical projection with a rounded tip. The apparatus was set up to apply a force of 0–200 mmHg, increasing from zero at a rate of 15 mmHg/s to the cutoff pressure of 200 mmHg. Mechanical paw pressure threshold was defined as the pressure to withdrawal of the hind paw. The mean baseline MPP threshold in the experiment was 60 mmHg (52–72 mmHg).

Motor function before and after intrathecal drug injection was examined using the scale proposed by Langerman et al. [18]for rabbits, which we applied to the rat model as follows: 0 = free movement of hind limbs without limitation; 1 = limited or asymmetrical movement of the hind limbs to support the body and walk; 2 = inability to support the back of the body on hind limbs, with detectable ability to move the limbs and respond to pain stimulus; and 3 = total paralysis of the hind limbs.

Intrathecal drug administration was accomplished by using a microinjection syringe (Hamilton, Reno, NV) connected to the intrathecal catheter in awake, briefly restrained rats. All drugs or drug combinations were administered manually over 10 s and followed by a 10-micro liter flush of sterile physiologic saline to ensure that the drug reached the spinal cord.

The drugs administered in the experiments were morphine hydrochloride (MW 375.85; Sankyo, Tokyo, Japan), omega-conotoxin GVIA (omega-CgTx, MW 3037; Research Biochemicals, Natick, MA), and naloxone hydrochloride (MW 363.84; Sankyo). Drugs were freshly dissolved in sterile physiologic saline in concentrations that allowed intrathecal injections in 10-micro liter volumes.

Behavioral assessments were performed between 10:00 and 12:00 AM to control for diurnal fluctuations in opioid sensitivity. Before the animals were used in each experiment, we measured the baseline TF latency, MPP threshold, and motor function to confirm the neurologically normal state. After determination of baseline values, rats received intrathecal injections of morphine (1, 2, 5, or 10 micro gram), omega-CgTx (0.05, 0.1, 0.25, or 0.5 micro gram), combination of morphine and omega-CgTx, or physiologic saline as control. Tail flick latencies or MPP thresholds were determined 5, 10, 15, 20, 30, 45, and 60 min after the intrathecal administration. In the animals receiving the combination of morphine and omega-CgTx, 200 micro gram/kg naloxone was administered intraperitoneally 61 min after administration of the combined drugs, and the latencies or thresholds were again evaluated 5 min after naloxone administration. The observers were not blinded as to which agent or combination was given each time.

The responses for the TF and MPP tests were calculated as the percent maximum possible effect (postdrug value - baseline value)/(cutoff value - baseline value) x 100. Dose-responses were constructed and compared using measurements at a set time in each animal (15 min after intrathecal administration).

Isobolographic analysis of interactions was used to define the nature of the functional interaction between morphine and omega-CgTx. From the dose-response curves of morphine and omega-CgTx alone, the respective dose producing a 50% maximum possible effect (ED⁵⁰value) was determined for each drug in both nociceptive tests to construct isobolographic analysis. When the morphine and omega-CgTx were coadministered, a constant dose ratio was used, based on the ED⁵⁰values of each agent administered separately so that equieffective doses were given together. The equieffective dose ratio was 20:1 for morphine and omega-CgTx, respectively, in both tests. Consequently, the doses of the combination of morphine and omega-CgTx were 0.3 micro gram and 0.015 micro gram, 0.5 micro gram and 0.025 micro gram, 1 micro gram and 0.05 micro gram, or 2 micro gram and 0.1 micro gram, respectively.

The effects of drugs on TF latency and MPP threshold were evaluated by one-way analysis of variance and Scheffe's F-test. A P value < 0.05 was considered to be statistically significant.

For evaluation of the interaction between intrathecal morphine and intrathecal omega-CgTx, isobolograms were constructed using ED⁵⁰values obtained when the agents were administered separately and together. The construction of the dose-response curves and the determination of doses producing 50% maximum possible effect (ED⁵⁰) as well as 95% confidence intervals were computed. The ED⁵⁰values and 95% confidence intervals for omega-CgTx and morphine alone were plotted on the X and Y axes, respectively, and the theoretical additive point was calculated according to the method described by Tallarida et al. [19]From the dose-response curve of the combined drugs, the ED⁵⁰value of the total dose of the combination was calculated, and based on the known dose ratio (20:1), the single doses of the agents in the combination were obtained for plotting on the isobologram. Statistical significance between the theoretical additive points and the experimentally derived ED⁵⁰value was evaluated using Student's t test. An experimental ED⁵⁰that was significantly less than the theoretical additive ED⁵⁰(P < 0.05) was considered to indicate a synergistic interaction between morphine and omega-CgTx.

The method of total fractions was calculated to obtain the magnitude of the interaction as described by Roerig et al. [20]Total fraction values were calculated as follows:

(ED⁵⁰of morphine when injected with omega-CgTx)/(ED⁵⁰of morphine alone)+(ED⁵⁰of omega-CgTx when injected with morphine)/(ED⁵⁰of omega-CgTx alone)

Values near 1 indicate an additive interaction, values less than 1 indicate a synergistic interaction, and values greater than 1 imply an antagonistic (less than additive) interaction between intrathecal morphine and omega-CgTx.

Intrathecally administered omega-CgTx alone produced a significant prolongation of TF latency and a significant increase in MPP threshold in a time- and dose-dependent manner (Figure 1and Figure 2). Peak effects of omega-CgTx were observed 15 min after intrathecal administration in both nociceptive tests. At a high dose of 0.5 micro gram omega-CgTx, most animals showed intermittent spontaneous TF and shaking behavior approximately 30–60 min after the administration, which limited the study on the measurement of TF latency at this period. However, at doses of 0.05, 0.1, and 0.25 micro gram omega-CgTx, the spontaneous TF and shaking behavior were not observed during 60 min after administration. Intrathecally administered morphine alone also showed antinociceptive effects in TF and MPP tests in a time-dependent manner (data not shown). The times to peak effects of intrathecal morphine in both tests were 15 min after the administration, which were same as the time observed to peak effects of intrathecal omega-CgTx. Figure 2shows the dose response curves for intrathecal morphine and omega-CgTx in the TF and MPP tests, which indicate that both drugs produced a dose-dependent antinociceptive effects in thermal and mechanical nociceptive tests.

In motor function testing, the scale before omega-CgTx administration was 0 and the scale after the administration at doses employed was 0 during the observation period (data not shown).

(Figure 3) shows the effects of an intrathecal ineffective dose of morphine (2 micro gram) combined with a slightly effective dose of omega-CgTx (0.1 micro gram) in the TF and MPP tests. In contrast to the ineffectiveness of 2 micro gram morphine and slight effectiveness of 0.1 micro gram omega-CgTx, the concomitant administration of the drugs produced a significant prolongation of the TF latency and a significant increase in MPP threshold. This potentiation was immediately reversed by intraperitoneally administered naloxone 200 micro gram/kg (Figure 3). Figure 4demonstrates the effects of the combination of morphine and omega-CgTx at the dose of the 20:1 ratio. In both nociceptive tests, these combinations demonstrate a dose- and time-dependent prolongation of the latency and increase in the threshold. At the combination doses employed in the experiment, the animals did not show any spontaneous TF or shaking behavior seen in the most animals treated with 0.5 micro gram omega-CgTx. The motor function scales were all 0 before and after the administration of the combined drugs (data not shown).

(Table 1) shows the ED⁵⁰s and 95% confidence intervals for morphine, omega-CgTx, and the combination in TF and MPP tests. As shown in Figure 5, the experimentally derived ED⁵⁰and 95% confidence interval in the isobologram decreased below the theoretically additive dose line. The confidence intervals of the theoretical additive point and those of the experimental point did not overlap in the isobolograms of TF and MPP tests. These analyses revealed a significantly synergistic interaction between morphine and omega-CgTx in TF and MPP tests (P < 0.05). The total fraction values, which indicate the magnitude of the interaction, were less than 1 in both tests, indicating a synergistic interaction (Table 1).

The current study demonstrated that intrathecally administered omega-CgTx produced antinociceptive effects against thermal and mechanical noxious stimuli in a time- and dose-dependent manner. We also demonstrated in this study that intrathecally coadministered morphine and omega-CgTx showed a synergistic antinociceptive interaction against both noxious stimuli.

The distinction between N- and L-channels is based on their different inactivation characteristics and their different pharmacologic properties; L-channels are blocked by dihydropyridine calcium blockers, [21]and N-channels, by omega-CgTx. [21,22]Some studies on the distribution of these two channel subtypes in sensory neurons show that L-channels may not be very common in nerve terminals and may be preferentially localized in the cell body, whereas N-channels are localized in the transmitter release zone in nerve terminals. [2]Thus, the N-, rather than L-type, is believed to be the most important channel in regulatory neurotransmitter release from the nerve terminals in the central nervous system.

N-type calcium channels are selectively blocked by omega-CgTx via a competitive mechanism. [23,24]The presence of specific omega-CgTx-binding sites was shown in the dorsal half of the rat spinal cord, especially superficial lamina of the dorsal horn (I and II of Rexed). [11,12,25]Magi et al. [10]reported that omega-CgTx significantly reduced the potassium-induced release of neuropeptides (substance P and CGRP) from the dorsal half of the rat spinal cord. Santicioli et al. [11]also demonstrated that omega-CgTx, but not L-type calcium channel blocker nifedipine, abolished an outflow of CGRP-like immunoactivity, which was evoked by electrical field stimulation from perfused slices of the dorsal half of the rat spinal cord. Thus, omega-CgTx sensitive N-type calcium channel opening must be essential for the triggering of neurotransmitter release from primary afferents. Therefore, disruption of calcium ion movement through N-type calcium channels by omega-CgTx and consequent inhibition of releasing neurotransmitters should interfere with normal sensory processing, resulting in contribution to antinociception. It seems that the dose-dependent antinociceptive action of intrathecally administered omega-CgTx shown in the current study reflects the inhibiting neurotransmitter release caused by the N-type calcium channel blockade.

Other less selective N-type calcium channel blockers such as aminoglycoside antibiotics, when administered intraperitoneally, produce analgesia in rats and mice as assessed by TF, carrageenan-induced articular incapacity, and hot plate tests. [26,27]Recently, Malmberg and Yaksh [28]demonstrated that spinal injection of certain synthetic omega-conopeptides, selective blockers for N-type voltage-dependent calcium channels, produced a powerful dose-dependent suppression of the first and second phase of the formalin test, although they only produced a modest antinociceptive effect to the hot plate test. Furthermore, in the model of peripheral nerve injury, omega-conopeptides produced a dose-dependent blockade of tactile allodynia. [29]Thus, it seems clear that the N-type calcium channel is involved in facilitated nociceptive processing at the spinal level in the models in which there is a hyperalgesic component.

A previous study showed that intracerebroventricular and intraperitoneal administration of large doses of omega-CgTx caused neurologic dysfunction (e.g., paroxysmal shaking, convulsion) and motor impairment. [30]In the current study, a large dose of intrathecal omega-CgTx (0.5 micro gram) caused intermittent spontaneous TF and shaking, which limited the measurement of TF latency 30–60 min after drug administration. The mechanism of these behaviors is unknown. The peak antinociceptive effects in TF and MPP tests were observed 15 min after administration, and the behavioral effects were not apparent at this time. Thus, it is unlikely that the spontaneous TF and shaking found in rats account for the suppression of nociceptive responses. We think that the changes in motor tone do not contribute to the suppression of nociceptive responses. Conversely, in motor function testing, the scores before and after the administration of omega-CgTx, morphine, and the combination were all 0 points, indicating lack of a loss of motor tone by omega-CgTx.

The most important result obtained in this study was that intrathecal N-type calcium channel blockers synergistically potentiated the antinociception of the intrathecal morphine. To investigate the antinociceptive interaction between intrathecal morphine and omega-CgTx, isobolographic analysis was adopted in the current study. Using this analysis, the results in this experiment revealed a significant synergistic antinociceptive interaction between morphine and omega-CgTx in the TF and MPP tests. The magnitude of the interaction, e.g., total fraction values, also less than 1, indicate a synergistic interaction. Furthermore, the degrees of synergism in TF and MPP tests were comparable.

Although the mechanism of the synergism was not clear, the observed synergy in the current study suggests that morphine and omega-CgTx act, in part, at distinct and separate sites to produce antinociception. Malmberg and Yaksh [31]reported that chronic infusion of omega-conopeptides that block N-type calcium channels produced a powerful antinociception, with minimal development of tolerance. In contrast, morphine infusion, which acts through a G protein-coupled channel, developed tolerance. This emphasizes the likelihood that the N-type channel blockers act in a manner separate from an opioid receptor.

Spampinato et al. [32]found that the antinociceptive responses elicited by intracerebroventricular micro-opioid agonists were significantly prolonged and potentiated by omega-CgTx. Morphine possesses the micro- and delta-agonistic activities to produce suppression of the responses to somatic nociception. [33]The activation of micro- and delta-receptors by a variety of ligands produces an opening of potassium ion channels, not directly, but via either cyclic adenosine monophosphate and/or G protein intermediations. [34,35]The net result of this action on a neuron would be hyperpolarization and a reduction in firing. Thus, opioid action through the micro and/or delta receptor might contribute to produce synergistic antinociception.

We demonstrated previously that L-type calcium channel blockers, verapamil, diltiazem, and nicardipine, also potentiated the antinociceptive effect of morphine at the spinal level. [8]Therefore, it seems that morphine produces synergistic antinociceptive interaction with N- and L-type calcium channel blockers. However, it is extremely difficult to interpret the molecular events underlying the behavioral effects induced by drug treatment because they are the result of the complex interaction and integration of different neuronal pathways.

In conclusion, intrathecally administered omega-CgTx produced antinociception in TF and MPP tests, emphasizing the importance of N-type voltage-dependent calcium channels in the spinal cord on nociception. The synergistic antinociceptive interaction observed after coadministration of omega-CgTx and morphine is suggestive of functional interaction between N-type voltage-dependent calcium channel and opioid receptor activation involved in nociceptive processing.