The enhancement of endotoxin-induced nitric oxide production by elevation of glucose concentration in macrophage

The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) are known to be modulated by a variety of factors. Recent study showed that endotoxin-induced NO synthesis and iNOS expression were greatly enhanced by elevation of extracellular glucose concentration in murine macrophages. Although this was suggested to be due to the activation of protein kinase C (PKC) via sorbitol pathway, there was lack of evidence for this speculation. This study was performed to delineate the underlying intracellular mechanisms of glucose-enhancing effect on endotoxin-induced NO production in Raw264.7 macrophages. The levels of NO release induced by lipopolysaccharide (LPS) significantly increased by the treatment of glucose in a concentration dependent manner and also, this effect was observed in LPS-preprimed cells. Concurrent incubation of cells with PKC inhibitors, H-7 or chelerythrine, and LPS resulted in the diminution of NO production regardless of glucose conc entration but this was not in the case of LPS-prepriming, that is, chelerythrine showed a minimal effect on the glucose- enhancing effect PMA, a PKC activator, did not show any significant effect on glucose-associated NO production. Modulation of sorbitol pathway with zopolrestat, an aldose reductase inhibitor, did not affect LPS-induced NO production and iNOS expression under high glucose condition. And also, sodium pyruvate, which is expected to normalize cytosolic NADH/NAD+ ratio, did not show any significant effect at concentrations of up to 10 mM. Glucosamine marginally increased the endotoxin-induced nitrite release in both control and high glucose treated group 6-diazo-5-oxonorleucine (L-DON) and azaserine, glutamine: fructose-6-phosphate amidotransferase (GFAT) inhibitors, significantly diminished the augmentation effect of high glucose on endotoxin-induced NO production. On the other hand, negative modulation of GFAT inhibitors was not reversed by the treatment of glucosamine , suggesting the minimal involvement, if any, of glucosamine pathway in glucose-enhancing effect. In summary, these results strongly suggest that the hexosamine biosynthesis pathway and the activation of PKC via sorbitol pathway do not contribute to the augmenting effect of high glucose on endotoxin induced NO production in macrophage-like Raw264.7 cells.

The Study of the Effect of Nerve Growth Factor on Intestinal Peristalsis in Streptozotocin-Induced Diabetic Rats / 대한해부학회지

Recently, it has been postulated that diabetic autonomic neuropathy is caused by reduction in availability of nerve growth factor (NGF) in enteric nervous system. This experiments were performed to determine the changes of the distribution of enteric neuropeptide by diabetes and these changes could be prevented by administration of NGF. Sprague Dawley rats (200~250gm) were made diabetic by a single intraperitoneal injection of streptozotocin 65 mg/kg in saline. Recombinant human NGF (Sigma, Co., Ltd.) were administered at a dose of 500ng/kg subcutaneously every day for consecutive 4 weeks after streptozotocin administration. After 4 weeks, rats were anesthetized with ether and perfused with 4% paraformaldehyde. ileum was dissected and prepared by whole mount preparation method. Prepared segments were immunostained for substance p, calcitonin gene-related peptide, vasoactive intestinal peptide, and galanin by PAP technique. For the observation of the interstitial cells of Cajal, segments were immersed in Champy-Maillet solution for 2 days Results obtained were as follows: 1. In myenteric plexus of diabetic rats, substance P-like and VIP-like immunoreactivity were not changed compared with that of the control group. CGRP-like and galanin-like immunoreactivity were decreased in diabetic group and immunoreactive cells for CGRP and galanin were also decreased 18.1% (P<0.01) and 43.7% (P<0.01) respectively. 2. In NGF administerd diabetic group, immunoreactivity of substance p, VIP, galanin in myenteric plexus were slightly increased and immunoreactive cells for substancre p, VIP, galanin were almost the same as that of the control group. However, immunoreactive cells for CGRP of myenteric plexus were not changed by NGF. 3. In submucous plexus of diabetic rats, immunoreactivity of all four neuropeptides(substance p, CGRP, VIP, galanin) were decreased compared with that of the control group. Immunoreactive cells for substance p, CGRP, VIP, and galanin were also decreased in 38.8%, 77.6%, 33.0%, and 35.7%, respectively (P<0.01). 4. In NGF administered diabetic group, immunoreactivities of substance p, VIP and galanin in submucous plexus were increased and the immunoreactive cells were increased significantly compared to diabetic group. However, immunoreactive cells for CGRP of submucous plexus were not changed by NGF. 5. Interstitial cells of Cajal of diabetic group were decreased 7.4% ovoidal cells (A type) and 28.3% round cells (B type) In NGF administered group, the morphology and the number of ICC were not different to the control group. With the above results, it could be assumed that NGF prevent the damage of neurotransmitter and ICC in enteric nervous system.

The Immunohistochemical and Morphometric Study of the Effect of Nerve Growth Factor on Spinal Ganglia in Streptozotocin-Induced Diabetic Rats / 대한해부학회지

Recently diabetic neuropathy has been postulated to occur from reduced availability of neurotrophic factor. This experiment was performed to identify the effect of nerve growth factor on dorsal root ganglia (DRG) in the strepto-zotocin-induced diabetic rat using morphometry and immunohistochemistry. The results obtained are as follows : 1. Unlike in the diabetic group where the type A and B cells were significantly decreased in their total numbers and sizes, these cells were normal in NGF-administered diabetic group. 2. Numbers of cells immunoreactive with SP and CGRP were also significantly decreased in the diabetic group. However, the NGF-administered diabetic group did not show any reduction in the number of these cells. 3. Mean sizes of cells immunoreactive with SP and CGRP cells were reduced in the diabetic group by 18.1% and 26.6% respectively (P<0.01). On the other hand, in NGF-administered diabetic group, mean sizes of SP-immunoreactive cells were increased (10.5%) which was not statiatically significant, and those of CGRP-immunoreactive cells were decreased (18%) compared to the control group (P<0.01). 4. In the diabetic group, many of nerve cell bodies showed some degenerative characteristics including neuron-satellite cell interface of irregular shape, the presence of a number of vacuoles and dense bodies, and nucleus of irregular contour. However, NGF-administered diabetic group exhibited neuron-satellite cell interface of regular form, many neurofilaments and neurotubules, and normal intracellular organelles. These results suggest that administration of NGF protects spinal ganglion cells from morphometric and morphological changes which are associated with a streptozotocin -induced diabetic neuropathy.

Cardioprotective effects of low dose bacterial lipopolysaccharide may not be directly associated with prostacyclin production

Sublethal dose of bacterial lipopolysaccharide (LPS) would induce protection against cardiac ischemic/ reperfusion (I/R) injury. This study examines the following areas: 1) the temporal induction of the cardioprotection produced by LPS; and 2) the relations between a degree of protection and the myocardial prostacyclin (PGI2) production. Rats were administered LPS (2 mg/kg, i.v.), and hearts were removed 1, 4, 8, 14, 24, 48, 72, and 96 h later. Using Langendorff apparatus, haemodynamic differences during 25 min of global ischemia/30 min reperfusion were investigated. The concentration of PGI2 in aliquots of the coronary effluent was determined by radioimmunoassay as its stable hydrolysis product 6-keto-PGF1alpha and lactate dehydrogenase release were measured as an indicative of cellular injury. LPS-induced cardiac protection against I/R injury appeared 4 h after LPS treatment and remained until 96 h after treatment. PGI2 release increased 2-3 fold at the beginning of reperfusion compared to basal level except in hearts treated with LPS for 48 and 72 h. In hearts removed 48 and 72 h after LPS treatment, basal PGI2 Was increased. To determine the enzymatic step in relation to LPS-induced basal PGI2 production, we examined prostaglandin H synthase (PGHS) protein expression, a rate limiting enzyme of prostaglandin production, by using Western blot analysis. LPS increased PGHS protein expression in hearts at 24, 48, 72, 96 h after LPS treatment. Induction of PGHS expression appeared in both isotypes of PGHS, a constitutive PGHS-1 and an inducible PGHS-2. To identify the correlationship between PGI2 production and the cardioprotective effect against I/R injury, indomethacin was administered in vivo or in vitro. Indomethacin did not inhibit LPS-induced cardioprotection, which was not affected by the duration of LPS treatment. Taken together, our results suggest that PGI2 might not be the major endogenous mediator of LPS-induced cardioprotection.

Effects of iontophoretically applied naloxone, picrotoxin and strychnine on dorsal horn neuron activities treated with high frequency conditioning stimulation in cats

Transcutaneous electrical nerve stimulation(TENS), acupuncture-needling, and electroacupuncture are useful non-ablative methods in medical practice for relief of pain. These procedures appear to work by causing an increased discharge in afferent nerve fibers which in turn modifies the transmission of impulses in pain pathways. It is known that the mechanism of analagesic effect via these maneuvers are variable depending on the stimulating parameters. For example, the endogenous opioid system is profoundly related to the mechanism when a peripheral nerve stimulation is applied with parameters of low frequency and high intensity. However, when stimulated with parameters of high frequency and high intensity, the reduced activity of dorsal horn neurons is only slightly reversed by a systemic administration of naloxone, a specific opiate antagonist. Thus, the present study was performed to investigate the neurotransmitter that concerns the mechanism of peripheral nerve stimulation with parameters of high frequency and high intensity. We used an iontophoretic application of antagonists of possible related neurotransmitters. The dorsal horn neuron activity which was evoked by squeezing the peripheral cutaneous receptive field, was recorded as an index of pain with a microelectrode at the lumbo-sacral spinal cord. Naloxone, picrotoxin and strychnine were applied at 200nA during a period of conditioning nerve stimulation. We observed the effects of these drugs on the change of dorsal horn neuron activities. The main results of the experiment can be summarized as follows. The spontaneous activity of dorsal horn neurons increased in the presence of glutamate and decreased with GABA. It did not change with naloxone, picrotoxin or strychnine. When naloxone was applied iontophoretically during peripheral nerve stimulation, there was no statistically significant analgesic effect compared with that of the control group. When picrotoxin was applied iontophoretically during peripheral nerve stimulation, the analgesic effect was reduced. When strychnine was applied, the analgesic effect was reduced but did not show a statistically significant difference with the control group. These results suggested that the GABAergic system may have been partially related in the analgesic action of peripheral nerve stimulation with parameters of high frequency and high intensity.

Mechanism of transmission and modulation of renal pain in cats; effect of nucleus raphe magnus stimulation on renal pain

Initially, when periaqueductal gray (PAG) is electrically stimulated, analgesia is induced, and this phenomenon is called stimulation-produced analgesia. Nucleus raphe magnus (NRM) as well as PAG are known to be the potent analgesic centers. NRM could modulate the nociceptive response of spinal cord neurons through spinally projecting fibers. However, as well as the above analgesic effects have been confined to the somatic pain, it was variable according to species, and the analgesic effect by NRM stimulation on the visceral pain was not yet clarified. In this study the analgesic effect by NRM stimulation on the visceral pain was examined through recording the activities of the dorsal horn neurons with renal input and renal pain, as a type of visceral pain. The renal pain was induced by ureteral occlusion or renal arterial occlusion, which in turn activated the renal mechanoreceptor or chemoreceptor. These cells had concomitant somatic input. In order to compare the effects of NRM stimulation on the renal pain with somatic pain, the somatic stimulation such as squeezing was conducted on the peripheral receptive field. The main results are summarized as follows: 1) After an electrical stimulation of NRM, spontaneous activities of dorsal horn neurons with renal input were reduced to 73.3 +/- 9.7% of the control value. 2) After an electrical stimulation of NRM, activities of dorsal horn neurons with renal input evoked by a brush, a type of non-noxious stimuli, did not change significantly. But the activities by a squeeze, a type of noxious stimuli, the activities were reduced to 63.2 +/- 7.2% of the control value. 3) After an electrical stimulation of NRM, activities of dorsal horn neurons with renal input evoked by occlusion of ureter or renal artery were reduced to 46.7 +/- 8.8% and 49.0 +/- 8.0% of the control value respectively. 4) The inhibitory effect of NRM on the dorsal horn neurons with renal input did not show any difference between renal A delta fiber and C fiber group. 5) By the electrical stimulation of NRM, the activities evoked by ureteral occlusion showed more reduction in the high threshold cell group than in the wide dynamic range cell group. These results suggest that activation of NRM can alleviate the renal pain as well as the somatic pain by modulating the dorsal horn neurons activities.

Effect of peripheral nerve stimulation on the dorsal horn cell activity in cats with cutaneous inflammation

There are some reports showing that an experience of long-enduring pain causes a change in the pain transmission system, suggesting a plastic nature of the nociceptive system. However, most of the studies concerning the analgesic effect of peripheral nerve stimulation dealt with normal animal or human subjects. So, the present study was undertaken to investigate the effect of peripheral nerve stimulation on the dorsal horn cell activity using a tonic pain model, which was made by producing a cutaneous inflammation. The main results are summarized as follows. 1) The evoked activity by electrical or natural stimulation as well as spontaneous activity was enhanced, and the receptive field size was also expanded by the inflammation. 2) Peripheral nerve conditioning stimulation reduced the C-response of the dorsal horn cell in the normal and inflamed group, and the degree of inhibition between the two groups showed no significant difference. 3) Inhibition of the C-response of the dorsal horn cells by peripheral conditioning stimulation was completely reversed by naloxone in the inflamed group whereas there was a partial block in the normal group.