Increase in participation of vasoactive intestinal peptide in relaxation of the distal colon of Wistar rats with age.

Changes in participation of vasoactive intestinal peptide (VIP) in nonadrenergic noncholinergic (NANC) relaxation of longitudinal muscle of the distal colon with age were studied in 2- to 50-week-old Wistar rats in vitro. The extent of the VIP-mediated component of the relaxation induced by electrical field stimulation (EFS) was determined by the effect of VIP(10 - 28), a VIP receptor antagonist. In 2-week-old rats, the extent of the VIP-mediated component of the relaxation was scarce, about 10%, whereas the component gradually increase with age and reached the maximum extent 66% at 50-week-old. Since our previous results suggest that VIP induces NANC relaxation via activation of charybdotoxin (ChTx, a blocker of large conductance Ca(2+)-activated K(+) channel)-sensitive K(+) channels with concomitant slow hyperpolarization in the muscle cells, we next studied whether ChTx-sensitive component and slow hyperpolarization changes with age. Extent of ChTx-sensitive component of the relaxation increased with age, showing a very similar pattern to VIP-mediated one. EFS induced monophasic inhibitory junction potentials (i.j.ps) in longitudinal muscle cells of the distal colon of 2- and 4-week-old. EFS also induced biphasic i.j.ps in many longitudinal muscle cells of 8- and 50-week-old: rapid and subsequent slow hyperpolarization. A VIP receptor antagonist selectively inhibited the slow hyperpolarization. Exogenously added VIP induced no appreciable change in the membrane potential of longitudinal muscle cells of 2-week-old, whereas it induced slight slow hyperpolarization of the cell membrane in 4-week-old and magnitude of the hyperpolarization increased with age. On the other hand, relaxant response of the longitudinal muscle to exogenously added VIP was high in younger rats. The present results suggest that the role of VIP in mediating NANC relaxation of longitudinal muscle of the Wistar rat distal colon is very little at neonatal stage, but it increases with age.

Differences in mediator of nonadrenergic, noncholinergic relaxation of the distal colon between Wistar-ST and Sprague-Dawley strains of rats.

Participation of nitric oxide and vasoactive intestinal peptide (VIP) in electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle and in balloon distension-induced descending NANC relaxation of circular muscle were studied in the distal colon of Wistar-ST and Sprague-Dawley rats. The extent of the nitric oxide-mediated component was approximately 50% in longitudinal and circular muscle of Sprague-Dawley rats, whereas this component was absent in both muscles of Wistar-ST rats. The extent of the VIP-mediated component was approximately 40% in longitudinal muscle of Wistar-ST rats and circular muscle of Sprague-Dawley rats, whereas this component was absent in circular muscle of Wistar-ST rats and longitudinal muscle of Sprague-Dawley rats. In circular muscle of Sprague-Dawley rats, in which participation of both nitric oxide and VIP in the relaxation was suggested, inhibition of descending relaxation by N(G)-nitro-L-arginine (L-NOARG) together with VIP-(10-28) was similar to that by either of the antagonists, and exogenous VIP-induced relaxation was not affected by L-NOARG, but exogenous nitric oxide-induced relaxation was partly inhibited by VIP-(10-28). These results suggest a linkage of the pathways mediated by nitric oxide and VIP. In the immunohistochemical studies, nitric oxide synthase or VIP immunoreactive neurons were seen in the ganglia, primary internodal strands of the myenteric plexus and in the circular muscle layer. However, the overall appearance of immunoreactive cell bodies in the myenteric plexus and the numbers of immunoreactive fibers in the circular muscle layer appeared to be similar in Wistar-ST and Sprague-Dawley rats. These results suggest that mediators of NANC relaxation in the distal colon are different in different strains of rats, i.e., Wistar-ST and Sprague-Dawley, although no such difference was seen in immunohistochemical studies.

Mediators of nonadrenergic, noncholinergic relaxation in longitudinal muscle of the intestine of ICR mice.

Mediators of nonadrenergic, noncholinergic (NANC) relaxation in longitudinal muscle of several regions of ICR mouse intestine were studied. An inhibitor of synthesis of nitric oxide, N(G)-nitro-L-arginine (L-NOARG) at 10 microM significantly inhibited NANC relaxations induced by electrical field stimulation (EFS) in the jejunum, ileum, and the proximal and distal colon. Especially in the ileum extent of the inhibition was more than 80%. An antagonist of vasoactive intestinal peptide (VIP) receptors, VIP(10-28) at 3 microM partially inhibited the EFS induced relaxations in the jejunum and proximal colon, but very slightly in the distal colon and had no effect in the ileum. An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptor, PACAP(6-38) at 3 microM partially inhibited the EFS-induced relaxations in the proximal and distal colon, but not in the jejunum and ileum. Totals of the percentages of relaxant components mediated by nitric oxide, VIP and PACAP in every region are roughly equal to a hundred percent. In another series of experiments, EFS-induced relaxations were almost completely inhibited by the treatment of the segments with L-NOARG and VIP(10-28) in the jejunum, with L-NOARG, VIP(10-28) and PACAP(6-38) in the proximal colon, and with L NOARG and PACAP(6-38) in the distal colon. The present results suggest that nitric oxide solely mediates the relaxation of longitudinal muscle of the ileum of ICR mice, whereas nitric oxide and VIP co-mediate it in the jejunum, nitric oxide, VIP and PACAP in the proximal colon, and nitric oxide and PACAP in the distal colon.

Postnatal development of NK1, NK2, and NK3 neurokinin receptors expression in the rat retina.

The biological effects of tachykinins are mediated by three distinct receptors, the neurokinin 1 receptor (NK1-R), NK2-R, and NK3-R. There is no information available concerning the development of these receptors in the retina. In the present study, we investigated the localization of tachykinin receptors, using antisera directed against NK1-R, NK2-R, and NK3-R in the adult and developing rat retinas. Numerous NK1-R immunoreactive (NK1-R IR) cells were already observed in the proximal part of the neuroblastic layer in the retina at postnatal day 5 (P5). The distribution and intensity of NK1-R IR cells and processes in the inner nuclear layer (INL) and inner plexiform layer (IPL) at P10 were similar to those of adult retina. Most NK1-R IR cells located in the proximal part of INL, which were morphologically amacrine cells. In the contrast to the early expression of NK1-R IR cells, no NK3-R IR structures existed in the neuronal elements of the retina until P10. NK3-R IR processes were first detected in the outer plexiform layer (OPL) at P10. At P15, NK3-R IR somata were slightly stained in the distal and middle parts of the INL, and NK3-R IR processes were present in the OPL and the upper part of the IPL. During P15-P30, the number of NK3-R IR somata located in the INL remarkably increased. These NK3-R IR cells were morphologically bipolar and amacrine cells. This study provides differential cellular distribution of NK1-R IR cells and NK3-R IR cells in the INL of the rat retina. Our findings suggest that NK1-R and NK3-R are involved in different visual circuits and retinal maturation, and NK3-R may play previously unknown important roles in the visual processes of the rat.

Enhanced interstitial expression of caldesmon in IgA nephropathy and its suppression by glucocorticoid-heparin therapy.

BACKGROUND: With progressive renal disease, structural derangement increasingly encompasses the tubulointerstitial compartment. Tubulointerstitial injury is a critical determinant of renal functional reserve and prognosis in renal disease. Interstitial cells acquiring characteristic of myofibroblasts are an important contributor to interstitial fibrosis. Caldesmon, a calmodulin or actin binding protein, is a molecular marker of differentiation in smooth muscle cells and has recently been shown by us to be a good marker of mesangial cell activation in IgA nephropathy patients. METHODS. We studied whether the expression of caldesmon in interstitium of the kidney was enhanced in the process of glomerular disease and whether it would be a marker of interstitial activation in specific disease states. We performed immunohistochemical staining with anti-caldesmon antibodies in 38 biopsy specimens from IgA nephropathy patients and analysed them quantitatively with a computer-aided manipulator. Interstitial caldesmon expression were compared with histological changes and clinical parameters. RESULTS: Caldesmon expression was enhanced where interstitial cell infiltration and fibrosis were found. Immunoelectron microscopy revealed that caldesmon staining in the renal interstitium was cytoplasmic, and in the processes of myofibroblast-like cells. Caldesmon expression was more prominent in the intense CD68 infiltrated group than in the low positive cells infiltrated group. Patients showing high intensity of interstitial caldesmon expression had significantly higher urinary protein excretion than those showing low intensity of caldesmon expression. Next, 15 patients were treated with glucocorticoid and heparin for 4-8 weeks and re-biopsies were performed. Caldesmon expression was reduced in concomitant with decreased interstitial cell infiltration. Follow-up of these patients (average 24 months) revealed a significant suppression of urinary protein excretion and significant improvement of creatinine clearance. CONCLUSION: These results suggest that the interstitial caldesmon expression is associated with the progression of IgA nephropathy, and glucocorticoid--heparin therapy may reverse the phenotypic change of interstitial cells during the disease process of glomerulonephritis.

150-kDa oxygen-regulated protein (ORP150) suppresses hypoxia-induced apoptotic cell death.

To determine the contribution of 150-kDa oxygen-regulated protein (ORP150) to cellular processes underlying adaptation to hypoxia, a cell line stably transfected to overexpress ORP150 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORP150 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 was maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense-transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, compared with ORP150 antisense transfectants and wild-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well recognized inducer of apoptosis in HEK cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wild-type and antisense cells. These data indicate that ORP150 has an important cytoprotective role in hypoxia-induced cellular perturbation and that ORP150-associated inhibition of apoptosis may involve mechanisms distinct from those triggered by other apoptotic stimuli.

Decrease in participation of nitric oxide in nonadrenergic, noncholinergic relaxation of rat intestine with age.

Participation of nitric oxide in the electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation in various intestinal regions was studied in 2- to 50-week-old Wistar rats. In the jejunum of 2-week-old rats, the extent of the nitric oxide-mediated component of the relaxation of longitudinal muscle was approximately 60-70%, whereas the component was 40-50% in 4-week-old rats and was absent in 8- and 50-week-old rats. Thus, nitric oxide seems to be the most important mediator at young ages but its significance is lost with age. The same tendency as that in the jejunum was also shown in longitudinal muscle of the ileum, proximal and distal colon, and rectum. The tendency was also shown in the circular muscle of the rectum. Sensitivity of the longitudinal muscle of the jejunum and proximal colon to exogenously added nitric oxide was high in younger rats. Immunoreactive structures for nitric oxide synthase were observed in the circular muscle layer of the rectum. The population of the structures was denser in 4-week-old than that in 50-week-old. The results suggest that NANC relaxation in every region of the intestine at 2-week-old is almost solely mediated by nitric oxide, and its significance as an inhibitory mediator gradually or rapidly decreases with age.

Nonadrenergic, noncholinergic relaxation mediated by nitric oxide with concomitant change in Ca2+ level in rectal circular muscle of rats.

The mediators of nonadrenergic, noncholinergic (NANC) relaxation of the circular muscle of rat rectum were examined in vitro. In the circular muscle of rat rectum, NG-nitro-L-arginine (L-NOARG) at 10 microM did not affect electrical field stimulation-induced relaxation but at 100 microM it inhibited electrical field stimulation-induced relaxation by about 75% and 1-mM L-arginine reversed the inhibition. Exogenous nitric oxide (NO) (1-10 microM) concentration dependently relaxed the circular muscle. Electrical field stimulation increased the cyclic GMP content of the circular muscle to about twice its resting level. L-NOARG, even at 10 microM, completely inhibited the electrical field stimulation-induced elevation of cyclic GMP content. However, L-arginine at 1 mM did not reverse the inhibition in cyclic GMP content. Inhibitory junction potentials (i.j.ps) induced by electrical field stimulation in the circular muscle cells were not affected by L-NOARG, 100 microM. Apamin ( < or = microM) did not affect the electrical field stimulation-induced relaxation, but almost completely inhibited electrical field stimulation-induced i.j.ps. NO (0.3-10 microM) induced relaxation of the circular muscle with a concomitant decrease in intracellular Ca2+ level ([Ca2+]i). Abundant immunoreactivity of NO synthase was found in the circular muscle layer, in addition to myenteric and submucosal plexus. The results suggest that NO induces NANC relaxation with a concomitant change in [Ca2+]i in the circular muscle of rat rectum. However, the involvement of changes in cyclic GMP level and in membrane potentials in the mechanism was not shown in the present experimental conditions.

A relationship between substance P receptor and retinal fibers in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) in the hypothalamus controls many of the circadian rhythms in mammalian species. In the present study, we investigated the location of substance P receptor (SPR)-containing neurons in the rat SCN, using a specific antibody against SPR, which corresponds to the NK-1 subtype of tachykinin receptors, and also examined the synaptic relationship between SPR-containing neurons and retinal fibers at the ultrastructural level. An SPR-immunoreactive meshwork of labeled somata and dendrites was identified in the SCN. The strongest SPR-immunoreactivity was observed in the dorsal and lateral parts of the SCN. Many labeled somata were identified there and their dendrites protruded ventrally from their somata. A few SPR-immunoreactive somata were observed also in the ventral part of the SCN and within the optic tract. In the SCN of eye-enucleated animals, degenerating retinal fibers were shown to terminate on SPR-immunoreactive dendrites forming asymmetrical axo-dendritic contacts.

Postnatal development of the substance P-, neuropeptide Y- and serotonin-containing fibers in the rat suprachiasmatic nucleus in relation to development of the retino-hypothalamic projection.

The suprachiasmatic nucleus (SCN) in the hypothalamus controls many of the circadian rhythms in mammalian species. In the present study, we investigated the development of substance P (SP)-, neuropeptide Y (NPY)- and serotonin (5-hydroxytryptamine, 5-HT)-immunoreactive fibers in the rat SCN and the development of the retino-hypothalamic tract using cholera toxin beta subunits (CTB), in order to understand which parts of the SCN participate in diurnal rhythm regulation and entrainment. In newborn rats, SP-, NPY- and 5-HT-immunoreactive fibers were scarcely detected in the SCN. The number of SP-immunoreactive fibers gradually increased between postnatal days (P) 15 and 30. At P30, the distribution pattern of SP-immunoreactive fibers in the SCN was similar to that in the adult rat. The number of NPY- and 5-HT-immunoreactive fibers increased greatly between P10 and P15, and the increase in NPY- and 5-HT-immunoreactive fibers continued until P20. CTB was injected into the unilateral eyeball of the rat at various postnatal stages. In neonates, several labeled retinal fibers already existed in the ventral part and ventro-lateral edge of the SCN. The number and density of labeled retinal fibers in the SCN gradually increased between P10 and P20. Between P20 and P30, a decrease in the labeling was observed in the dorsolateral part of the SCN. The adult pattern of labeled retinal fibers was achieved between P20 and P30. The development of SP-immunoreactive fibers was delayed about 10 days relative to that of NPY-, 5-HT-immunoreactive fibers and retinal fibers.

c-fos expression after formalin injection into the face in the cat.

Expression of the c-fos gene in CNS induced by formalin injection into the face as pain stimulus was examined in cats. Fos-positive neurons were demonstrated in the anterior cingulate, anterior insula and other areas of the cerebral cortex bilaterally, midline thalamic nuclei, hypothalamus, and brainstem in both the formalin-injected and control group (anesthesia only). Most of these labeled regions appeared to correspond to stress- and anesthesia-related sites. The difference from the control was the finding of Fos-positive cells in the SI, possible SII and trigeminal subnucleus caudalis in the experimental group. Almost significant increase in Fos-positive cells was also observed in areas 24, 23 and the anterior agranular insular cortex in experimental cats. Our findings appear to be compatible with recently reported PET findings in man, except laterality. For full appreciation of the complex pain experience, these diverse areas of the brain appear to be activated.

A novel family of heparin-binding growth factors, pleiotrophin and midkine, is expressed in the developing rat cerebral cortex.

We raised specific antibodies to the pleiotrophin and the midkine gene product, which comprise a novel heparin-binding growth factor family. Immunocytochemistry using these antibodies revealed that both proteins were strongly expressed in the developing cerebral cortex in the rat in distinct fashion. The midkine gene product was primarily detected in the embryonic period and was not observed in the postnatal cerebral cortex, whereas pleiotrophin was most intense in the early postnatal period, in addition to being present the embryonic period. Pleiotrophin was also localized in the adult cerebral cortex, albeit with much less intensity than in the embryonic and early postnatal period. These temporal patterns were consistent with those of the mRNA expression. The ultrastructural localization of both factors showed markedly similar profiles; immunopositive substances were primarily associated with the cell surface and were found preferentially in the regions where cell migration and neurite outgrowth take place, implying that this family functions primarily in developmental events mediated by cell-cell contact. The present results suggest that the midkine gene product and pleiotrophin are differentially involved in cortical development at different stages.

Projections of tachykinin- and glutaminase-containing rat retinal ganglion cells.

Glutamate (Glu) and the tachykinin substance P (SP) have been proposed as neurotransmitters or neuromodulators of the retinal projection to the brain. In the present study, we demonstrate that tachykinin-like (TK) immunoreactivity (IR) accumulates in rat retinal axons following electrical lesions to the optic tract, indicating that SP is conveyed in the optic nerve to its central targets. In addition, we show that eye enucleation causes a dramatic decrease in TK-IR fibers in the pretectal olivary nucleus (PON), but not in other retinorecipient nuclei of the thalamus and the midbrain, and that Fluorogold injected into the pretectum is retrogradely transported to the somata of TK-IR retinal ganglion cells (RGCs), indicating an important projection of TK-IR RGCs to the PON. We also show that most rat RGCs are labeled with antibodies against phosphate-activated glutaminase, an enzyme considered to generate the transmitter pool of glutamate. Unlike TK-IR fibers, phosphate-activated glutaminase-IR structures disappear in most retinorecipient nuclei following eye enucleation. The present results give neuroanatomical support to the idea that glutamate is a neurotransmitter in the retinal projection and suggest an important role for TK-IR RGCs in the relay of visual information to the PON.

Differential effects on substance P immunoreactivity in rat suprachiasmatic and olivary pretectal nuclei.

We examined the effects of constant dark or constant light on substance P (SP) and/or neuropeptide Y (NPY) immunoreactive fibres in the rat suprachiasmatic nucleus (SCN) and olivary pretectal nucleus (OPT) by immunohistochemistry. After constant dark, SP immunoreactive fibres and terminals decreased slightly in the SCN, while they increased markedly in the OPT. After constant light, they increased markedly in the SCN, but were little changed in the OPT. NPY immunoreactive fibres and terminals in the SCN decreased slightly after after constant light, but there were no effects on these fibres after constant dark. These findings suggest that SP immunoreactive fibres are involved in mediating illumination discrimination in the SCN and/or OPT.

Substance P-immunoreactive innervation from the retina to the suprachiasmatic nucleus in the rat.

The present study examined substance P (SP) innervation in the suprachiasmatic nucleus (SCN) of the rat. In the colchicine-untreated rat, SP-immunoreactive fibers formed a dense oval plexus in the ventral part of the SCN. After bilateral eye enucleation, there was a marked reduction in SP-immunoreactive fibers in the ventral part of the SCNs. The SP-immunoreactive neurons in the retinal ganglion cell layer were retrogradely labeled after injection of Fluoro-gold into the SCN. These findings indicate the presence of the SP innervation from the retina to the SCN in the rat. The role of SP in the retino-hypothalamic tract was discussed from the light-dark cycle.

Ultrastructure and retinal innervation of deafferentation-induced enkephalin-immunoreactive elements in the superficial layers of the rat superior colliculus.

Leu-enkephalin-like immunoreactive (ENK-I) elements appearing in the superficial layers of the rat superior colliculus (SC) after eye-enucleation were examined by means of immunoelectronmicroscopy. ENK-I somata were of a single type and formed symmetric and asymmetric synapses with non-immunoreactive axon terminals. Some degenerating retinal terminals made synaptic contacts only with small ENK-I dendrites, suggesting that deafferentation-induced ENK-I neurons in the rat SC receive retinal input onto the distal portions of their dendrites.

Retinal fibers make synaptic contact with neuropeptide Y and enkephalin immunoreactive neurons in the intergeniculate leaflet of the rat.

Direct synapses between retinal fibers and neuropeptide Y (NPY) and/or enkephalin-immunoreactive (ENK-I) neurons were investigated in the intergeniculate leaflet (IGL) of the rat, using combined immunohistochemical and degeneration techniques. Degenerating retinal fibers terminated on dendrites of NPY-I and/or ENK-I neurons. NPY-I fibers in the IGL formed axo-dendritic synapses on non-immunoreactive dendrites. Most of them were asymmetrical synapses. ENK-I axon terminals contacted non-immunoreactive dendrites with symmetrical synapse.

Striatal calcitonin gene-related peptide-like immunoreactive afferents from the regions ventral and medial to the medial geniculate nucleus of rats.

The sources of fibers with immunoreactivity to calcitonin gene-related peptide in the caudal portion of the caudate-putamen were investigated in animals treated by a knife cut, a tracer injection, or an electrolytic lesion of regions ventral and medial to the medial geniculate nucleus. Hemitransection of the brain just caudal to the caudate-putamen decreased ipsilaterally calcitonin gene-related peptide-like immunoreactivity in the caudal portion of the caudate-putamen, suggesting that calcitonin gene-related peptide-like immunoreactive fibers in the caudate-putamen originated from neurons in the area caudal to the caudate-putamen. Some calcitonin gene-related peptide-like immunoreactive neurons in the regions ventral and medial to the medial geniculate nucleus, including the peripeduncular, posterior intralaminar and suprageniculate nuclei, were labeled ipsilaterally with a retrograde tracer after an injection into the caudal portion of the caudate-putamen. In addition, an electrolytic lesion of calcitonin gene-related peptide-like immunoreactive neurons in the regions ventral and medial to the medial geniculate nucleus decreased the number of calcitonin gene-related peptide-like immunoreactive fibers in the ipsilateral caudal portion of the caudate-putamen. These results suggest that some cells containing calcitonin gene-related peptide in several posterior thalamic nuclei project to the caudal caudate-putamen.

Effects of eye-enucleation on substance P-immunoreactive fibers of some retinorecipient nuclei of the rat in relation to their origin from the superior colliculus.

We have previously shown that retinal deafferentation causes a decrease in immunoreactive dendrites of substance P-positive neurons of the superficial superior colliculus of the rat. Since some retinorecipient thalamic and pretectal nuclei are putative targets for substance P-containing cells of the superior colliculus, the present study attempted to ascertain whether substance P-immunoreactive fibers in these nuclei are also affected by retinal denervation. We found that unilateral eye removal produced a progressive increase in fibrous substance P immunoreactivity in the nucleus of the optic tract, lateral posterior nucleus, and lateral geniculate nucleus of the side contralateral to the enucleation. On the other hand, unilateral lesions to the superficial layers of the superior colliculus produced a dramatic reduction in substance P immunoreactivity in the ipsilateral nucleus of the optic tract, lateral posterior nucleus, and dorsal and ventral lateral geniculate nuclei. In bilaterally enucleated animals, unilateral lesion to the superior colliculus produced, as expected, loss of immunoreactive fibers only in the lateral posterior nucleus and the retinorecipient nuclei ipsilateral to the lesion. These results suggest that transneuronal changes in the distribution of substance P in collicular neurons observed after enucleation could be reflected in their projections to the other primary visual centers and to the lateral posterior nucleus.