Changes in responsiveness of freshly isolated longitudinal muscle cells from rat uterus towards oxytocin during gestation: contractility and calcium signaling.

Changes in responsiveness of freshly isolated longitudinal muscle cells from rat uterus to oxytocin during gestation were investigated through measuring contractility as well as intracellular free calcium concentration. We have demonstrated the pregnant stage-dependent contraction of freshly isolated myometrial cells in response to an extracellular hormone, oxytocin, in Ca2+-containing medium. The oxytocin effect appeared to be through oxytocin receptor since the effect could be blocked by a specific oxytocin antagonist. The magnitude of the contraction of the isolated cells in response to extracellular oxytocin was in the order of 21 day >> 18 day > 15 day pregnant rat longitudinal muscle cells. In a concentration dependent manner, oxytocin elicited a rapid increase in [Ca2+]i of longitudinal muscle cells isolated from different stages of the pregnant rat uterus, especially at the term of pregnancy. The time (4-5 s) required to reach a maximum increase in [Ca2+]i of the isolated longitudinal muscle cells in response to oxytocin was the shortest among all previously reported studies. The results also indicated that the freshly prepared longitudinal muscle cells maintained their functional calcium signaling system. The order of the responsiveness of the isolated longitudinal muscle cells to oxytocin was 21 day >> 18 day > 15 day pregnant rats in terms of rate, affinity and magnitude. Oxytocin appears to transmit its signal mainly through stimulating a voltage-dependent and/or receptor operated nonselective calcium channel. However, the possibility that a part of the oxytocin action occurs through stimulating the release of calcium from intracellular store sites of longitudinal muscle still remains.

Ultrastructural findings of the endothelial cells in pancreatic lesions after chronic administration of methamphetamine in rats.

The effects of chronic administration of methamphetamine on the pancreas were studied using electron micrography in an experimental model. Methamphetamine (1 mg/kg/day) was subcutaneously injected in five-week-old male Wistar Kyoto rats (WKY) for 8 weeks. Five age and sex-matched WKY served as controls. In light microscopy, scattered necrosis, intercellular vacuolization and severe hemorrhage were the prominent lesions in the methamphetamine-treated rats. The associated ultrastructural alterations consisted of degenerated, swollen, mitochondria with disrupted cristae and cell debris in the superficial space of the endothelial cells, and extensive vacuolization in the degenerated endothelial cells of small vessels and in the smooth muscle cells of their wall. However, in this study, hypercontration or lamellar membrane-like changes were not identified. It is suggested that pancreatic necrotic hemorrhage is a consequence of methamphetamine damage to endothelial cells in various organs, including pancreas.

A histopathological study of pancreatic lesions after chronic administration of methamphetamine to rats.

The effects of chronic administration of methamphetamine on pancreatic tissues were histopathologically studied in experimental models. Methamphetamine (1 ml/kg body weight/day) was subcutaneously injected into 14 five-week-old male Wistar Kyoto rats (WKY) for 12 weeks. Age and sex matched 5 WKY rats served as controls. With light microscopy, some scattered edematous lesions and moderate vacuolization were demonstrated in the pancreas of 8 of the methamphetamine treated rats. However, in 4 of the rats, severe regional hemorrhage, partial acinal cell necrosis, destruction of the acinal cells, neutrophile infiltration, interstitial vessel dilatation, interstitial edema and fatty cell invasion were observed after the injections of methamphetamine. In 2 other animals, fibrosis and cirrhosis-like lesions with destruction and degeneration of the acinal cells were observed the small vessels had a slight degeneration of the endothelial cells. In the control animals, no lesions, except for some edematous lesions were found. In all cases, there were no nesidioblastosis-like lesions or necrosis of the Langerhans's islets. In the immunohistochemical study using anti- alpha 1-chymotrypsin antibody, more positive reactive cells were demonstrated among the interstitial and inter acinal cells, both in number and degree, in the methamphetamine treated rats. In addition to the animal model, there were 4 autopsy cases of sudden death in chronic methamphetamine abusers. The autopsies demonstrated a severe acute necrotic hemorrhagic pancreatitis, with only scattered slight hemorrhaging in the brain and lungs. These findings indicate that chronic administration of methamphetamine to rats evoked significant changes in pancreatic tissues including some degeneration of the endothelial cells of the small vessels in this hypoxia-vulnerable organ.

Genetic and histologic evidence implicates role of inflammation in traumatic brain injury-induced apoptosis in the rat cerebral cortex following moderate fluid percussion injury.

Traumatic brain injury (TBI) causes massive brain damage. However, the secondary injury and temporal sequence of events with multiple mechanisms after the insult has not been elucidated. Here, we examined the occurrence of apoptosis and a causal relationship between inflammation and apoptosis in the TBI brain. Following a lateral moderate fluid percussion injury model of TBI in adult rats, microarray analyses detected apparent changes in the expression levels of apoptosis-related genes which revealed time-dependent expression patterns for 23 genes in the lateral cortex. The upregulated 23 genes included inflammatory cytokines such as interleukin 1 (IL-1) α, IL-1ß, and tumor necrotic factor (TNF) which immediately increased at 3 h following the injury. Time-dependent gene expression profile analyses showed that apoptosis was subsequently induced following inflammation. These results taken together suggested changes in expression of apoptosis-related genes may be associated with inflammatory response. Accompanying this surge of cell death genes after TBI was a neurostructural pathologic hallmark of apoptosis characterized by leakage of cytochrome c into cytoplasm, DNA fragmentation and apoptotic cells in the lateral cortex of the impacted hemisphere. Caspase-3 positive cells in the TBI brain were initially sporadic after 3 h, but these apoptotic cells subsequently increased and populated the cerebral cortex at 6 and 12 h, and gradually reached a plateau by 48 h. Interestingly, the expression profile of CD68 macrophage labeled cells closely resembled that of apoptotic cells after TBI, including the role of inflammatory signaling pathway in the progression of apoptotic cell death. These results taken together suggest that TBI induced upregulation of apoptosis-related genes, concomitant with the detection of apoptotic brain pathology during the 3-48 h post-injury period, which may be likely mediated by inflammation. Therapies designed at abrogating apoptosis and/or inflammation may prove effective when initiated at this subacute TBI phase.

Oxytocin-induced phosphorylation of myosin light chain is mediated by extracellular calcium influx in pregnant rat myometrium.

Studies of oxytocin-induced phosphorylation of myosin light chain (MLC), resulting in myometrial contraction, suggest that extracellular Ca(2+) influx is involved in its signal transduction. To explore the possibility that intracellular Ca(2+) mobilization by oxytocin may also contribute to MLC phosphorylation, we investigated the relative contributions of these Ca(2+) sources to oxytocin signal transduction in myometrium of pregnant rat. In pregnant rat myometrium, oxytocin-induced Ca(2+) influx occurs via an L-type voltage-dependent Ca(2+) channel. Treatment with verapamil, an antagonist specific for these channels, significantly diminished MLC phosphorylation observed in response to oxytocin administration without affecting the release of Ca(2+) from intracellular Ca(2+) stores. Furthermore, oxytocin-induced MLC phosphorylation was not observed when extracellular Ca(2+) was not present. Our results clearly indicate that extracellular Ca(2+) influx, rather than release from Ca(2+) storage sites, is essential for oxytocin-induced MLC phosphorylation.

Characterization and expression of oxytocin and the oxytocin receptor.

Oxytocin, a nonapeptide hormone and neurotransmitter, is expressed in a variety of tissues, as are its receptors. In vivo, oxytocin acts as a paracrine and/or autocrine mediator of multiple biological effects. These effects are exerted primarily through interactions with G-protein-coupled oxytocin/vasopressin receptors, which, via G(q) and G(i), stimulate phospholipase C-mediated hydrolysis of phosphoinositides. It is generally recognized that, during pregnancy, oxytocin plays a major role in increasing myometrial contractility at term, and that it acts on its cardiac receptor to decrease the cardiac rate and force of contraction. It is, however, doubtful that increased endocrine oxytocin concentration is involved in the onset and progression of normal human labor.

Effects of oxytocin, prostaglandin F2 alpha and prostaglandin E2 on intracellular free calcium concentrations of longitudinal muscle cells isolated from term pregnant rat myometrium.

The effects of oxytocin, PGF2 alpha and PGE2 on [Ca2+]i of isolated longitudinal muscle cells of term pregnant rats were investigated using Fura-2. Oxytocin, PGF2 alpha and PGE2 induced an initial rapid increase followed by a secondary gradual increase in [Ca2+]i in the presence of 1.5 mM Ca2+. The initial maximum increases in [Ca2+]i were obtained at 6 s for oxytocin, 20 s for PGF2 alpha and 30 s for PGE2 after addition of the stimulants. The EC50 values obtained from the curves were 2.0 nM for oxytocin, 250 nM for PGF2 alpha and 2,200 nM for PGE2. On the other hand, the increases in [Ca2+]i induced by the stimulants were nearly abolished by removal of the extracellular Ca2+. The stimulants induced biphasic increases in [Ca2+]i which were highly extracellular Ca(2+)-dependent processes, and the order of potencies for the stimulants was oxytocin > > PGF2 alpha > PGE2 in terms of affinity as well as magnitude. These results might indicate differences in the weight of the physiological role of the stimulants for regulating uterine contractility.

Effects of recombinant nitrophorin-2 nitric oxide complex on vascular smooth muscle.

Nitrophorin-2, isolated from the salivary gland of the blood-sucking insect Rhodnius prolixus, is a nitric oxide (NO) binding protein. We investigated the effects of recombinant nitrophorin-2 NO complex on vascular smooth muscle. The course of relaxation was relative to released NO from recombinant nitrophorin-2 NO complex. Our data suggested nitrophorin-2 was tightly adhesive to the membranes to transport NO into the cell during the insect sting.