Hydrogen sulfide as a novel mediator for pancreatic pain in rodents.

OBJECTIVE: Hydrogen sulfide (H(2)S) is formed from l-cysteine by multiple enzymes including cystathionine-gamma-lyase (CSE) in mammals, and plays various roles in health and disease. Recently, a pronociceptive role for H(2)S in the processing of somatic pain was identified. Here, the involvement of H(2)S in pancreatic pain is examined. METHODS: Anaesthetised rats or mice received an injection of NaHS, a donor for H(2)S, 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 Ca(2+) 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/H(2)S most probably targets T-type Ca(2+) channels, leading to nociception, and that endogenous H(2)S produced by CSE and possibly T-type Ca(2+) channels are involved in pancreatitis-related pain.

Luminal hydrogen sulfide plays a pronociceptive role in mouse colon.

OBJECTIVE: Given recent evidence that hydrogen sulfide (H(2)S), a gasotransmitter, promotes somatic pain through redox modulation of T-type Ca(2+) channels, the roles of colonic luminal H(2)S in visceral nociceptive processing in mice were examined. METHODS: After intracolonic administration of NaHS, an H(2)S donor, visceral pain-like behaviour and referred abdominal allodynia/hyperalgesia were evaluated. Phosphorylation of extracellular signal-regulated protein kinase (ERK) in the spinal dorsal horn was determined immunohistochemically. The whole-cell recording technique was used to evaluate T-type Ca(2+) currents (T-currents) in cultured dorsal root ganglion (DRG) neurons. RESULTS: Like capsaicin, NaHS, administered intracolonically at 0.5-5 nmol per mouse, triggered visceral nociceptive behaviour accompanied by referred allodynia/hyperalgesia in mice. Phosphorylation of ERK in the spinal dorsal horn was detected following intracolonic NaHS or capsaicin. The behavioural effects of intracolonic NaHS were abolished by a T-type channel blocker or an oxidant, but not inhibitors of L-type Ca(2+) channels or ATP-sensitive K(+) (K(ATP)) channels. Intraperitoneal NaHS at 60 micromol/kg facilitated intracolonic capsaicin-evoked visceral nociception, an effect abolished by the T-type channel blocker, although it alone produced no behavioural effect. In DRG neurons, T-currents were enhanced by NaHS. CONCLUSIONS: These findings suggest that colonic luminal H(2)S/NaHS plays pronociceptive roles, and imply that the underlying mechanisms might involve sensitisation/activation of T-type channels probably in the primary afferents, aside from the issue of the selectivity of mibefradil.

[Multiple goals in conflict resolution: their antecedents and effects upon tactical preference].

The multiple goals theory of conflict management (Ohbuchi & Tedeschi, in press) postulated that participants in a conflict pursue to achieve resource goals (economic and personal resources) and social goals (relationship, identity, justice, and power-hostility). The hypotheses based on this theory were examined by the episode method, in which 207 university students were asked to rate their recent experiences of interpersonal conflicts in terms of participants' attributes, goals, and tactics. More than 80% of the subjects answered that they were motivated to achieve multiple goals in their attempts to resolve the conflicts. Social goals were found to be more strongly activated, and economic resource goals were least strongly activated. Regression analyses revealed that the effects of participants' attributes on tactical preference were mediated by goals.

Phenotypic characteristics and significance of reticular meshwork surrounding splenic white pulp of mice.

This report describes the phenotypic characteristics of the reticular meshwork (RM) localized between the marginal zone and white pulp of mice. Considered from an anatomical point of view, RM contributes to the migration of lymphocytes into splenic white pulp. We divided the RM into two parts: one surrounding the periarterial lymphoid sheaths (PALS) and the other surrounding the follicles. Throughout both locations of RM, alkaline phosphatase activity was noted on the plasma membrane of the reticular cells and tenascin was expressed on the collagen fibrils in the matrix of the reticular fibers. In contrast, the expression of MAdCAM-1, one of the homing receptors of lymphocytes, was solely confined to the plasma membranes of the fiber-forming reticular cells and processes of the perifollicular RM. In aly mice with defective perifollicular RM and loss of MAdCAM-1 expression, lymphocyte homing to the white pulp was remarkably inhibited, and the compartmentalization of T and B cells did not occur. This fact supports the notion that perifollicular RM plays an essential role in lymphocyte homing and compartmentalization.

The covering method: an improved negative staining method for ultrathin cryo-sections of tissue.

Ultrathin cryo-sections of biological tissues for electron microscopy provide numerous advantages in cytochemical and morphological studies. However, difficulties still remain in the application of negative staining to cryo-sections. This paper describes a method for negative staining of cryo-sections by covering the grid with a piece of either Collodion or Formvar film. By doing so, appropriate amount of staining solution is retained on the grid for positive effects. Also, the present method requires only a few seconds to stain the ultrathin cryo-sections while previously published methods took up to 10 min.

An ultrathin frozen section is suitable for demonstrating cytochrome c oxidase activity at electron microscopic level.

Cytochrome c oxidase activity in rat liver was demonstrated in 40 microns sections and ultrathin frozen sections (100-200 nm in thickness), using the diaminobenzidine method. Electron microscopic observations showed strong activity in 81.9 +/- 13.1% of mitochondria along the edge, and 29.9 +/- 21.2% in the center of 40 microns sections. On the other hand, all mitochondria possessed strong activity in ultrathin frozen sections under the same experimental condition. In the ultrathin frozen section, the mitochondrial membranes, as well as the plasma membrane, were broken. The present study indicates that the ultrathin frozen section is suitable for demonstrating cytochrome c oxidase activity at electron microscopic level.

Characterization of the functional stages of osteoclasts by enzyme histochemistry and electron microscopy.

To investigate the functional stages of osteoclasts, the ultrastructural histochemical distribution of the lysosomal enzymes [acid phosphatase (tartrate-sensitive) and neutral phosphatase], the plasma membrane enzymes [alkaline phosphatase, Ca(++)-ATPase, and alkaline ouabain-insensitive p-nitrophenylphosphatase (alkaline p-NPPase)], and the mitochondrial enzyme (cytochrome C oxidase) was evaluated in the chicken tibial metaphysis. Both active-appearing and detached (resting) osteoclasts were studied. Serial sectioning was used to identify detached osteoclasts which were present in the perivascular space. The ultrastructure of detached osteoclasts was similar to that of active osteoclasts, except for the lack of a ruffled border and clear zone, and an altered distribution pattern of small vesicles. Small vesicles were uniformly distributed in the cytoplasm of resting osteoclasts, whereas they were concentrated beneath the ruffled border of active osteoclasts. Alkaline p-NPPase, a marker enzyme for the basal ruffled border, was also apparent on the membrane of small vesicles. However, the vesicles did not possess Ca(++)-ATPase, a marker enzyme for the apical plasma membrane. These findings support the concept that small vesicles serve as a membrane reservoir for the ruffled border membrane. Pre-osteoclasts contained abundant mitochondria and lysosomes, prominent Golgi complexes, moderately developed endoplasmic reticulum, and lacked small vesicles. Pre-osteoclasts appear to fuse with osteoclasts which are attached to the bone surface, but not with detached osteoclasts. The small vesicles, from which the ruffled border arises, are absent from pre-osteoclasts, suggesting that they develop after fusion with pre-existing osteoclasts or after attachment to the bone surface. Alkaline p-NPPase appears to be a marker for differentiation of pre-osteoclasts to mature osteoclasts.

Ultrastructural localization of adenylate cyclase activity in chicken osteoclasts.

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.

Ultrastructural localization of tartrate-resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone.

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.

Ultrastructural localization of guanylate cyclase in bone cells.

Guanylyl imidodiphosphate (GMP-PNP) hydrolyzing enzyme activity as a means of detecting plasma membrane guanylate cyclase was demonstrated in osteoblasts of chicken tibial metaphysis using a lead citrate histochemical method at the electron microscopic level. Activity was not discerned in osteoclasts or osteocytes. The reaction product development was completely abolished when the sections were incubated with substrate-free or MnCl2-free medium. Guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP) was a less effective substrate than GMP-PNP, and Mn++ was a stronger stimulator than Mg++. No reaction product was observed on the plasma membrane of osteoblasts when beta-glycerophosphate or p-nitrophenylphosphate was used as substrate instead of GMP-PNP. The results implicate guanylate cyclase as a significant effector of osteoblast regulation at the site of the plasma membrane.

[Combination of self-reinforcement and external-reinforcement on matching-to-sample learning].

This study aimed at to investigate the combined effects of self-reinforcement (SR) and external-reinforcement (ER) on a matching-to-sample learning task. Four colored cards with four different marks (20, 10, 0, -10) were employed. Subjects of 27 college students were divided into three groups; SR, ER, and SR-ER groups, and they were requested to choose correct cards to score 20 marks in each trial. Subjects in SR and SR-ER groups were required to express their degrees of confidence by exhibiting either one, two or three chips of token accordingly. Following results were obtained. First, reinforcing powers were greater under both SR and SR-ER conditions than ER condition. Second, SR-ER group manifested a remarkably higher learning effect compared to SR group. This is interpreted as that the former established a sort of self-assurance due to ER contingency during this learning condition. Finally, the number of chips SR and SR-ER groups presented corresponded to the hitting rate of marks between n and n + 1 trials.

IgA nephropathy with subendothelial deposits.

IgA nephropathy with subendothelial deposits in the capillary walls of the glomeruli (IgA type 2) was compared histometrically and clinically with IgA nephropathy without subendothelial deposits (IgA type 1) and membranoproliferative glomerulonephritis with subendothelial deposits (MPGN). Study cases consisted of 32 biopsies from 26 patients of IgA type 1, 25 biopsies from 20 patients of IgA type 2 and 31 biopsies from 27 patients of MPGN. Histological changes of the glomeruli consisted of an increase in the mesangial matrix and hypercellularity in the mesangium in both types of IgA nephropathy, and the degree of the changes was a little higher in IgA type 2 than in IgA type 1 (0.02 < P < 0.05). Mesangial changes of MPGN were marked as compared with IgA type 1 and IgA type 2 (P < 0.001). Histometry of the mesangium on the cases followed up showed that the degree of mesangial thickening increased with lapse of time in IgA type 2 and MPGN, whereas it remained unchanged up to 13 years in IgA type 1. Proteinuria tended to be mild in IgA type 1, moderate in IgA type 2, and marked in MPGN. The impairment of renal function was observed in 21.9% of IgA type 1, in 36.0% of IgA type 2 and in 58.1% of MPGN. IgA type 2 has been shown to be pathologically and clinically intermediate between IgA type 1 and MPGN. These results suggest that there is a clinicopathological overlap between IgA nephropathy and MPGN with IgA deposition.