Yoshihisa Kamanaka

Hydrogen sulfide as a novel mediator for pancreatic pain in rodents

Objective: Hydrogen sulfide (H2S) is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE) in mammals, and plays various roles in health and disease. Recently, a pronociceptive role for H2S in the processing of somatic pain was identified. Here, the involvement of H2S in pancreatic pain is examined. Methods: Anaesthetised rats or mice received an injection of NaHS, a donor for H2S, or capsaicin into the pancreatic duct, and the expression of spinal Fos protein was detected by immunohistochemistry. Pancreatitis was created by 6 hourly doses of caerulein in unanaesthetised mice, and pancreatitis-related allodynia/hyperalgesia was evaluated using von Frey hairs. CSE activity and protein levels in pancreatic tissues were measured using the colorimetric method and western blotting, respectively. Results: Either NaHS or capsaicin induced the expression of Fos protein in the superficial layers of the T8 and T9 spinal dorsal horn of rats or mice. The induction of Fos by NaHS but not capsaicin was abolished by mibefradil, a T-type Ca2+ channel blocker. In conscious mice, repeated doses of caerulein produced pancreatitis accompanied by abdominal allodynia/hyperalgesia. Pretreatment with an inhibitor of CSE prevented the allodynia/hyperalgesia, but not the pancreatitis. A single dose of mibefradil reversed the established pancreatitis-related allodynia/hyperalgesia. Either the activity or protein expression of pancreatic CSE increased after the development of caerulein-induced pancreatitis in mice. Conclusions: The data suggest that pancreatic NaHS/H2S most probably targets T-type Ca2+ channels, leading to nociception, and that endogenous H2S produced by CSE and possibly T-type Ca2+ channels are involved in pancreatitis-related pain.

Mitochondrial impairment induced by poly(ADP-ribose) polymerase-1 activation in cortical neurons after oxygen and glucose deprivation

Neuronal cells injured by ischemia and reperfusion to a certain extent are committed to death in necrotic or apoptotic form. Necrosis is induced by gross ATP depletion or ‘energy crisis’ of the cell, whereas apoptosis is induced by a mechanism still to be defined in detail. Here, we investigated this mechanism by focusing on a DNA damage‐sensor, poly(ADP‐ribose) polymerase‐1 (PARP‐1). A 2‐h oxygen and glucose deprivation (OGD) followed by reoxygenation (Reox) induced apoptosis, rather than necrosis, in rat cortical neurons. During the Reox, PARP‐1 was much activated and autopoly(ADP‐ribosyl)ated, consuming the substrate, NAD+. Induction of apoptosis by OGD/Reox was suppressed by overexpression of Bcl‐2, indicating mitochondrial impairment in this induction process. Mitochondrial permeability transition (MPT), or membrane depolarization, and a release of proapoptotic proteins, i.e. cytochrome c, apoptosis‐inducing factor and endonuclease G, from mitochondria were observed during the Reox. These apoptotic changes of mitochondria and the nucleus were attenuated by PARP‐1 inhibitors, 1,5‐dihydroxyisoquinoline and benzamide, and also by small interfering RNA specific for PARP‐1. These results indicated that PARP‐1 plays a principal role in inducing mitochondrial impairment that ultimately leads to apoptosis of neurons after cerebral ischemia.

The potent inducible nitric oxide synthase inhibitor ONO-1714 inhibits neuronal NOS and exerts antinociception in rats

We evaluated if ONO-1714, known as an inducible nitric oxide synthase (iNOS) inhibitor, could inhibit neuronal NOS (nNOS) and exert antinociception. ONO-1714 potently inhibited both crude rat cerebellar NOS and recombinant human nNOS in vitro. Systemic ONO-1714 at 1-10 mg/kg suppressed carrageenan-induced thermal hyperalgesia in rats, an effect being equivalent to the antinociception caused by L-NAME or 7-nitroindazole at 25 mg/kg. The same doses of ONO-1714 also caused hypertension. Intrathecal (i.t.) ONO-1714 potently reduced the hyperalgesia, the effective dose range (0.2-0.6 microg/rat) being much lower than the antinociceptive dose (150 microg/rat) of i.t. L-NAME. Thus, ONO-1714 is considered a potent inhibitor of nNOS in addition to iNOS. The distinct relative antinociceptive activities of systemic and i.t. ONO-1714 are attributable to its possible poor blood-brain barrier permeability.