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Aims Neurotropin® (NTP), an analgesic for chronic pain, has antihyperalgesic effects in specific alternation of rhythm in temperature (SART)-stressed rats. Previous studies have shown that SART stress induces hyperalgesia, as well as post-translational modification of proteins (including substrates for calpain, a calcium-dependent cysteine protease) in the mesencephalon of rats. To better understand the mechanism of action of NTP, we investigated whether SART stress activates calpain in the mesencephalon of rats and whether NTP inhibits this activation. Main methods Wistar rats were exposed to SART stress for 5 days. NTP (200 NU/kg/day) was administered intraperitoneally every day from the onset of SART stress. The mechanical pain threshold was measured using the Randall-Selitto test on the 6th day. Thereafter, the rat mesencephalon was immediately collected and calpain activity was examined using western blot analysis with a calpain cleavage site-specific antibody. Key findings SART stress induced hyperalgesia and increased the calpain activity in the mesencephalon of rats. In contrast, NTP treatment attenuated the hyperalgesia and prevented the increase in calpain activity in the mesencephalon of SART-stressed rats. Interestingly, a negative correlation was identified between calpain activity and mechanical pain threshold in SART-stressed rats treated with or without NTP. Furthermore, NTP inhibited calpain activity on mammalian uncoordinated-18 in rat mesencephalon homogenate and Ac-LLY-AFC as substrates in an in vitro cell-free system. Significance Our data demonstrate that NTP treatment prevents SART stress-induced calpain activation in the mesencephalon of rats and suggests that NTP-mediated antihyperalgesia is associated with an inhibition of calpain activity in the mesencephalon.

Aims In this study, we investigated the combined effect of Neurotropin® and pregabalin for L5-spinal nerve ligation (L5-SNL) model in rats and thiopental-induced sleep in mice. Main methods The left fifth lumbar nerve of rats was tightly ligated with silk sutures under pentobarbital anesthesia. The hindpaw withdrawal threshold was measured by application of von Frey filaments. Thiopental sodium was intravenously administered in mice and sleeping time was measured. In L5-SNL rats, an isobolographic analysis was performed to clarify the combined antiallodynic effect of Neurotropin and pregabalin 14 days after ligation in rats. In isobolographic analysis and thiopental-induced sleep test, Neurotropin and pregabalin were orally administered to coincide with the timing of the peak effect of each drug. Key findings Neurotropin (50–200 NU/kg) and pregabalin (2.5–10 mg/kg) showed a dose-dependent antiallodynic action in L5-SNL rats. The antiallodynic effect of pregabalin was reversed by intrathecal injection of yohimbine or ondansetron. Isobolographic analysis suggested that the combined antiallodynic effect of Neurotropin and pregabalin in L5-SNL rats may have been more than a mere additive effect. Neurotropin (50–400 NU/kg) had no effect on thiopental-induced sleeping time whereas pregabalin (30–100 mg/kg) significantly prolonged it. When the dose of pregabalin was 30 mg/kg, Neurotropin (50–400 NU/kg) did not further exacerbate the prolongation effect of pregabalin on thiopental-induced sleep. Significance It was suggested that when Neurotropin was administered in combination with pregabalin, it might provide more effective pain relief than that obtained with each agent alone in neuropathic pain without aggravating adverse effects of pregabalin.

Aims Although the clinical use of Neurotropin ® as an analgesic for chronic pain has been firmly established, its analgesic mechanism is still unclear. In this study, we investigate the direct effects of Neurotropin using an electrophysiological method. Main methods Blind patch-clamp recordings were made from rat locus coeruleus (LC) and periaqueductal gray (PAG) neurons in brainstem slices of normal rats. The effects of intracerebroventricular (icv) injection of Neurotropin on nociceptive transmission were recorded from spinal substantia gelatinosa (SG) neurons in fifth lumbar spinal nerve-ligated (L5-SNL) rats using an in vivo patch-clamp method. Key findings Neurotropin (0.2–1.0 NU/mL) dose-dependently increased the firing rate in noradrenergic LC neurons of normal rats. Under the voltage-clamp condition, Neurotropin induced an inward current in 90% of LC neurons that was not affected by tetrodotoxin or an injection of GDP-β-S (G protein inhibitor) through recording pipettes. In contrast, Neurotropin had no effects on all PAG neurons tested. Using in vivo patch-clamp recordings, the icv injection of Neurotropin inhibited both frequency and amplitude of pinch-evoked excitatory postsynaptic currents of SG neurons in L5-SNL rats. These results suggest that Neurotropin directly excites the descending noradrenergic LC neurons and inhibits nociceptive transmission in the spinal dorsal horn. Significance This study is the first direct demonstration that Neurotropin activates the noradrenergic descending pain inhibitory systems, and this would reinforce the usefulness of Neurotropin in the treatment of human neuropathic pain.

Neurotropin, a non-protein extract from the inflamed skin of rabbits inoculated with vaccinia virus, has been clinically used as an analgesic drug in Japan. Its analgesic effect has been demonstrated by reduced mechano-nociception in hyperalgesic rats exposed to SART-stress (a repeated cold stress) for 5 days. In order to clarify the mechanism of the analgesic effect of neurotropin at the spinal cord level, we examined the effects of several neurotransmitter receptor antagonists given by intrathecal (i.t.) injection on the antinociceptive effect of intraperitoneally (i.p.) injected neurotropin [100 and 200 Neurotropin Unit (NU)/kg]. The analgesic effect of neurotropin was significantly inhibited not only by methysergide (100 nmol Rat , i.t.), a non-selective antagonist against serotonin (5-HT), but also MDL 72222 (30 nmol Rat , i.t.), a selective 5-HT3 antagonist, but not influenced by ketanserin (100 nmol Rat , i.t.), a 5-HT2A antagonist. The antinociceptive effect of neurotropin (200 NU Kg , i.p.) was significantly inhibited also by yohimbine (30 nmol Rat , i.t.), a noradrenergic α2 antagonist. However, the analgesic effect of neurotropin (100 and 200 NU Kg , i.p.) was not influenced by naloxone (30 nmol Rat , i.t.), an opioid antagonist. These results suggest that the mechanism of the antinociceptive effect of neurotropin is via enhancement of endogenous descending pain inhibitory pathways of the serotonergic and noradrenergic systems, especially involving 5-HT3 and noradrenergic α2 receptors in spinal dorsal horn in which these neurons terminate. No influence of opioid receptors at the spinal cord level is indicated.

In order to verify the contribution of right atrial pressure to atrial natriuretic polypeptides (ANP) release, we measured plasma levels of immunoreactive (ir)-ANP when graded rise of right atrial pressure was executed in anesthetized dogs. Increasing right atrial pressure (RAP) from 2.7 +/- 0.6 to 9.0 +/- 0.7 mmHg, plasma levels of ir-ANP in aorta tended to increase by 33% but not significantly (p greater than 0.05). However, when RAP was increased from 9.0 +/- 0.7 to 17.0 +/- 1.1 mmHg, ir-ANP levels in aorta were significantly (p less than 0.05) increased by 132% of control within 5 min from the start of RAP elevation. The RAP elevation produced a sustained increase in plasma levels of ir-ANP. There was a positive correlation between right atrial pressure and plasma levels of ir-ANP. The plasma levels of ir-ANP were similar between aorta and pulmonary artery. These results demonstrate that increasing atrial pressure is closely correlated with ANP release and ANP is not greatly metabolized by pulmonary circulation.