Il Suk Yang

Water soluble fraction (<10 kDa) from bee venom reduces visceral pain behavior through spinal α2-adrenergic activity in mice

We have previously shown that subcutaneous bee venom (BV) injection reduces visceral pain behavior in mice, but it is not clear which constituent of BV is responsible for its antinociceptive effect. In the present study, we now demonstrate that a water-soluble subfraction of BV (BVA) reproduces the antinociceptive effect of BV in acetic acid-induced visceral pain model. We further evaluated three different BVA subfractions that were separated by molecular weight, and found that only the BVAF3 subfraction (a molecular weight of <10 kDa) produced a significant antinociceptive effect on abdominal stretches and suppressed visceral pain-induced spinal cord Fos expression. Injection of melittin (MEL), a major constituent of BVAF3, also produced a visceral antinociception. However, melittins antinociception was completely blocked by boiling for 10 min at 100 degrees C, while boiling either whole BV or BVAF3 did not prevent their antinociception. The antinociceptive effect of BVAF3 was completely blocked by intrathecal pretreatment with the alpha2-adrenoceptor antagonist, yohimbine (YOH), while intrathecal pretreatment with the opioid antagonist, naloxone (NAL) or the serotonin antagonist, methysergide, had no effect. These data demonstrate that BVAF3 is responsible for the visceral antinociception of whole BV and further suggest that this effect is mediated in part by spinal alpha2-adrenergic activity.

Effect of Bee Venom and Its Melittin on Apical Transporters of Renal Proximal Tubule Cells

Renal failure by bee venom may be related to a malfunction of renal transporters. However, the effects of bee venom on apical membrane transporters of renal proximal tubular cells are not yet known. The aim of this study was to examine the effects of dried bee venom of Apis mellifera and its melittin on apical transporter activity of primary cultured rabbit kidney proximal tubule cells. Bee venom (1 μg/ml) decreased the cell viability and increased lactate dehydrogenase activity over 30–min treatments. Its effect was blocked by mepacrine or AACOCF3 (10–6 M; phospholipase A2 inhibitors). However, there was no effect on cell viability at a concentration of 0.01 μg/ml of bee venom. Thus, we investigated the effect of bee venom (1 μg/ml) on the activity of renal transporters at 30 min. Bee venom inhibited α–methyl–D–glucopyranoside, Pi, and Na+ uptakes, but increased Ca2+ uptake. These effects of bee venom were blocked by mepacrine or AACOCF3 (10–6 M), and bee venom–induced stimulation of Ca2+ uptake was also blocked by methoxyverapamil and nifedipine (L–type calcium channel blockers). In addition, bee venom increased [3H]–arachidonic acid release by 216 % of that of control. In all experiments, bee venom melittin (0.5 μg/ml) had an identical effect to that of bee venom itself. In conclusion, bee venom inhibited, in part, α–MG, Pi, and Na+ uptakes through its melittin which increased Ca2+ uptake and arachidonic acid release in primary cultured rabbit renal proximal tubule cells.

High Glucose Stimulates Ca2+ Uptake via cAMP and PLC/PKC Pathways in Primary Cultured Renal Proximal Tubule Cells

Alteration of [Ca²⁺]_i by hyperglycemia is implicated in the pathogenesis of diabetic nephropathy. However, the effect of high glucose on Ca²⁺ regulation in proximal tubule cells is not known. Thus, we examined the mechanisms by which high glucose regulates Ca²⁺ uptake in primary cultured rabbit renal proximal tubule cells. Glucose increased the Ca²⁺ uptake in a time– and dose–dependent manner. A stimulatory effect of high glucose on Ca²⁺ uptake is predominantly observed using 25 mM glucose (high glucose) after 1 h, while 25 mM glucose did not affect cell viability and lactate dehydrogenase release. However, 25 mM mannitol and L–glucose did not affect Ca²⁺ uptake as compared with controls. Nifedipine and methoxyverapamil (L–type Ca²⁺ channel blockers) blocked high–glucose–induced stimulation of Ca²⁺ uptake. High–glucose–induced stimulation of Ca²⁺ uptake was blocked by pertussis toxin, SQ–22536 (adenylate cyclase inhibitor), myristoylated amide 14–22 (protein kinase A inhibitor), neomycin and U–73122 (phospholipase C inhibitors), and staurosporine and bisindolylmaleimide I (protein kinase C inhibitors). In addition, KN–62 (a Ca²⁺/calmodulin–dependent protein kinase II inhibitor) and W–7 (a Ca²⁺/calmodulin antagonist) blocked high–glucose–induced stimulation of Ca²⁺ uptake. In conclusion, high glucose stimulates the Ca²⁺ uptake through L–type Ca²⁺ channels via G–protein–coupled adenylate cyclase/cAMP and phospholipase C/protein kinase C pathways.

Alteration of nitrergic neuromuscular transmission as a result of acute experimental colitis in rat

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4% acetic acid in the rat. Animals were sacrificed at 4 h and 48 h post-acetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nω-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acid-treated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.

Visceral Hypersensitivity and Altered Colonic Motility in Type 2 Diabetic Rat.

Background/Aims Abnormal visceral sensitivity and disordered motility are common in patients with diabetes mellitus. The purpose of the present study was to investigate whether visceral sensation and bowel motility were altered in a rat model of type 2 diabetes mellitus accompanied by weight loss. Methods A type 2 diabetic rat model in adulthood was developed by administrating streptozotocin (STZ; 90 mg/kg, i.p.) to neonatal rats. Eight weeks after STZ administration, rats with blood glucose level of 200 mg/dL or higher were selected and used as diabetic group (n = 35) in this study. Abdominal withdrawal reflex and arterial pulse rate were measured to examine visceral nociception induced by colorectal distension (0.1–1.0 mL). The amplitude, frequency, and area under the curve (AUC) of spontaneous phasic contractions of colonic circular muscles were recorded in vitro to examine colonic motility. Results STZ-treated diabetic rats gained significantly less weight for 8 weeks than control (P < 0.01). Forty-eight percent of the diabetic rats showed enhanced visceral nociceptive response to colorectal distension. Diabetic rats did not differ from control rats in colorectal compliance. However, the frequency and AUC, not the amplitude, of colonic spontaneous contraction in vitro was significantly decreased in diabetic rats compared to control rats (P < 0.01 in frequency and P < 0.05 in AUC). Conclusions These results demonstrate visceral hypersensitivity and colonic dysmotility in a rat model of type 2 diabetes mellitus accompanied by weight loss.