Interaction between optineurin and the bZIP transcription factor NRL.

Although the gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis, it is ubiquitously expressed in all body tissues, including the retina. To study the function of OPTN in retinal ganglion cells as well as the whole retina, we previously isolated OPTN-interacting proteins and identified the gene encoding the bZIP transcription factor neural retina leucine zipper (NRL), which is a causative gene for retinitis pigmentosa. Herein, we investigated the binding between OPTN and NRL proteins in HeLaS3 cells. Co-expression of HA-tagged NRL and FLAG-tagged OPTN in HeLaS3 cells followed by immunoprecipitation and Western blotting with anti-tag antibodies demonstrated the binding of these proteins in HeLaS3 cells, which was confirmed by proximity ligation assay. NRL is the first OPTN-binding protein to show eye-specific expression. A series of partial-deletion OPTN plasmids demonstrated that the tail region (423-577 amino acids [aa]) of OPTN was necessary for binding with NRL. Immunostaining showed that Optn (rat homologue of OPTN) was expressed in rat photoreceptors and localised in the cytoplasm of photoreceptor cells. This is a novel demonstration of Optn expression in photoreceptor cells. OPTN was not detected in photoreceptor nuclei under our experimental conditions. Further analyses are necessary to elucidate the function of OPTN and the significance of its possible binding with NRL in photoreceptor cells.

Differential modulation of immunostimulant-triggered NO production by endoplasmic reticulum stress inducers in vascular smooth muscle cells.

We investigated the effects of endoplasmic reticulum (ER) stress inducers thapsigargin (TG) and tunicamycin (Tm) on immunostimulant lipopolysaccharide/interferon (LPS/IFN)-induced expression of isoform of nitric oxide synthase (iNOS) and nitric oxide (NO) production in vascular smooth muscle cells. LPS/IFN-induced iNOS mRNA expression was markedly enhanced by TG, whereas iNOS mRNA expression was strongly attenuated by Tm. Similarly, production of iNOS protein was markedly upregulated by TG but virtually eliminated by Tm. LPS/IFN-induced guanosine triphosphate cyclohydrolase I mRNA expression was slightly reduced by TG and markedly inhibited by Tm. Similarly, LPS/IFN-mediated induction of cellular biopterin was modestly reduced by TG and markedly inhibited by Tm. TG modestly enhanced LPS/IFN-induced activation of NF-κB, whereas Tm had no effect on it. Cellular respiration was reduced by TG and Tm in a concentration-dependent manner, which was confirmed by apoptosis assay. Thus, TG and Tm-induced ER stress and differently modulated NO production through alterations in iNOS expression and activity independently of NF-κB activation and caused a similar degree of ER stress-induced apoptosis.

YPEL5 protein of the YPEL gene family is involved in the cell cycle progression by interacting with two distinct proteins RanBPM and RanBP10.

YPEL5 is a member of the YPEL gene family that is highly conserved in the eukaryotic species and apparently involved in a certain cell division-related function. In this study, we examined the functional and phylogenetic aspects of YPEL5 protein in more detail. During cell cycle, YPEL5 protein was detected at different subcellular localizations; at interphase, it was located in the nucleus and centrosome, then it changed location sequentially to spindle poles, mitotic spindle, and spindle midzone during mitosis, and finally transferred to midbody at cytokinesis. Knockdown of YPEL5 function by siRNA or anti-sense morpholino oligonucleotide inhibited the growth of cultured COS-7 cells and early development of medaka fish embryos, indicating its involvement in cell cycle progression. Interestingly, RanBPM (Ran Binding Protein in the Microtubule organizing center, encoded by RANBP9) was identified as a YPEL5-binding protein by yeast two-hybrid method. A paralog of RanBPM, namely RanBP10 (encoded by RANBP10), was found to be another YPEL5-binding protein, and these two protein genes are highly conserved each other. Comparative genomic analysis allowed us to define a new gene family consisting of RanBPM and RanBP10, named Scorpin, providing a basis to better understand how they interact with YPEL5.

Shiga toxin 2eB-transgenic lettuce vaccine: N-glycosylation is important for protecting against porcine edema disease.

Porcine edema disease (ED) is a life-threatening toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) in weaned piglets. We previously reported that the stx2eB-transgenic lettuce 2BH strain shows potential for use as an oral vaccine candidate against ED. However, the 2BH strain expressed a hemagglutinin (HA)-tag together with Stx2eB and contained non-canonical N-glycosylation. Therefore, we developed two Stx2eB-lettuce strains, the 3 (G+) strain in which the HA-tag was removed from 2BH, and the 3 (G-) lettuce strain, in which the 73rd Asn was replaced with Ser to prevent non-canonical N-glycosylation of Stx2eB from the 3 (G+) strain. We examined the protective effect of these newly developed two strains compared with the previous 2BH strain against ED using a colostrum-deprived piglet STEC infection model. We found that the N-glycosylated 2BH and 3 (G+) strains relieved the pathogenic symptoms of ED in STEC-challenged piglets, whereas the non-glycosylated 3 (G-) strain did not. N-Glycosylation of the Stx2eB product in lettuce may be involved in the immune response in piglets.

Shiga toxin 2eB-transgenic lettuce vaccine is effective in protecting weaned piglets from edema disease caused by Shiga toxin-producing Escherichia coli infection.

Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB-transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB-lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB-lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB-lettuce is a promising first oral vaccine candidate against ED.

Cilostazol activates AMP-activated protein kinase and restores endothelial function in diabetes.

BACKGROUND: Endothelial dysfunction plays a key role in atherogenesis. We investigated whether AMP-activated protein kinase (AMPK) activity is a downstream mediator of the beneficial effects of cilostazol on vascular endothelial cells and whether cilostazol might reverse endothelial dysfunction in diabetic rats. METHODS AND RESULTS: Treatment of human umbilical vein endothelial cells (HUVECs) with cilostazol resulted in time-dependent activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase (ACC). Activation of AMPK by cilostazol was through signaling pathway independent of cyclic AMP and caused phosphorylation of endothelial nitric oxide synthase (eNOS), leading to increased production of nitric oxide (NO), while inhibiting cytokine-induced nuclear factor-kappaB (NF-kappaB) activation, leading to suppression of VCAM-1 gene expression. Significantly reduced eNOS activity and NO production in response to cilostazol and attenuation of cilostazol-induced inhibition of NF-kappaB activation were observed in cells treated with AMPK siRNA. We also demonstrated that administration of cilostazol to diabetic rats significantly restored endothelium-dependent vasodilation. Furthermore, treatment of diabetic rats with cilostazol increased tetrahydrobiopterin (BH4) levels in the aorta. Thus, recovery of BH4 following administration of cilostazol might also contribute to restoration of endothelial function in diabetic rats. CONCLUSIONS: Our findings suggest that the beneficial effects of cilostazol on endothelial function may be due to AMPK activation. Restoration of endothelial dysfunction in diabetic rats by cilostazol is at least partly attributed to amelioration of biopterin metabolism in the aorta.

Oral administration of tetrahydrobiopterin slows the progression of atherosclerosis in apolipoprotein E-knockout mice.

OBJECTIVE: Although it has been reported that oral administration of tetrahydrobiopterin (BH4) prevents endothelial dysfunction and vascular oxidative stress in various rat models, the effect of treatment with BH4 on atherogenesis remains unclear. METHODS AND RESULTS: In this study, we investigated whether oral BH4 treatment might slow the progression of atherosclerosis using hypercholesterolemic apolipoprotein E-knockout mice. We report that ingesting BH4 in drinking water is sufficient to inhibit atherogenesis in mice. Furthermore, we report that BH4 treatment improves endothelial dysfunction and attenuates increased mRNA expression of NADPH oxidase components, as well as a number of inflammatory factors, such as LOX-1 and MCP-1, in the aortas of apolipoprotein E- knockout mice. CONCLUSION: Strategies such as oral administration of BH4 to ensure continuous BH4 availability may be effective in restoring nitric oxide-mediated endothelial function and limiting vascular disease and the progression of atherosclerosis.

Expression of Na(+)/K(+)-ATPase alpha subunit isoforms in rat salivary glands: occurrence of sense and antisense RNAs of the alpha3 isoform in the sublingual gland.

We examined the expression of Na(+)/K(+)-ATPase alpha-subunit isoforms in rat salivary glands by RT-PCR. Isoform alpha1 was expressed strongly in all three major salivary glands. The alpha2 isoform was expressed in both submandibular gland (SMG) and sublingual gland (SLG) but faintly in the parotid gland (PG). The alpha3 was detected only in the SLG, and the alpha3 mRNA in the SLG was 1/8 of its level in the brain. Na(+)/K(+)-ATPase alpha3 isoform in the SLG, was localized predominantly on the basolateral plasma membranes in serous cells by immunohistochemical method. We also found the presence of natural antisense RNA of the alpha3 isoform in rat SLG: the 1st-strand cDNA prepared with gene-specific forward primers targeted to the CDS region and to the promoter region of the alpha3 gene in place of oligo(dT) or gene-specific reverse primers produced reasonable PCR products corresponding to the alpha3 cDNA sequence by the subsequent PCR reaction. Synthesis of the 1st-strand cDNA with the gene-specific forward primers was prevented by RNase digestion of the total RNA preparation, indicating that the PCR products in the RT-PCR system were not due to the contaminated genomic DNA, if any. The alpha3 protein level in rat SLG increased with aging, and levels of both alpha3 mRNA (sense RNA) and alpha3 antisense RNA were higher in SLGs of aged rats than in those of young rats, respectively.

Decreasing traits of fecal immunoglobulin A in neonatal and weaning piglets.

The concentration of fecal secretory immunoglobulin A (sIgA) in neonate and weaning piglets was measured daily from 1 day after birth to 50 days of age. The concentration of fecal sIgA started from the level of 10(4) microg/g wet feces 1 day after birth and then increased to a maximal value of up to 10(5) microg/g within a few days of birth. The values constantly declined to between 10(1) and 10(2) microg/g for the next 10 days and were relatively constant until weaning. The level of sIgA in the feces remained very low until at least 50 days of age. The vulnerability of pre- or post-weaning piglets can be explained, at least in part, by this low level of sIgA in the intestine.

Buthionine sulfoximine causes endothelium dependent hyper-relaxation and hypoadiponectinemia.

A close relationship between oxidative stress, endothelial dysfunction, and hypoadiponectinemia has been observed. The present study was performed to investigate how glutathione depletion via buthionine sulfoximine (BSO) administration affects endothelial function and adiponectin levels in rats. Acetylcholine (Ach)-induced vasodilation was significantly enhanced in BSO-treated rats, compared with control rats. This was completely abolished by L-NAME, and Ach-induced vasodilation was not observed in the aorta without endothelium. These results suggest that Ach-induced hyper-relaxation of the aorta in BSO-treated rats is completely dependent on the presence of endothelium and mediated by changes in eNOS activity. Catalase significantly inhibited this relaxation to Ach and no effect of catalase on sodium nitroprusside-induced relaxation of the aorta without endothelium was observed in BSO-treated rats. Thus, hyper-relaxation of the aorta in BSO-treated rats is likely caused by H2O2 in addition to NO produced by the endothelium via an eNOS-dependent mechanism. Hypoadiponectinemia and decreased levels of adiponectin mRNA in adipose tissue were observed in BSO-treated rats. Protein expression of eNOS and SODs (SOD-1 and SOD-2) in the aorta was increased and plasma NOx levels were decreased in BSO-treated rats. Our results suggest that oxidative stress induced by BSO causes eNOS uncoupling and hyper-relaxation by producing H2O2, and that BSO-induced oxidative stress causes hypoadiponectinemia, probably by increasing H2O2 production in adipose tissue.

Evaluation of the low dose level of a heat-killed and dried cell preparation of Enterococcus faecalis to prevent porcine edema disease using experimental infection model with enterotoxcemic Escherichia coli in weaning pigs.

Porcine edema disease (ED) is caused by Shiga toxin 2e-producing Escherichia coli (STEC). ED has become frequent in pig farms, and the use of antimicrobials has resulted in the development of antimicrobial-resistant STEC. Accordingly, the use of materials other than antimicrobials is requested for the prevention of ED. Oral administration of a heat-killed and dried cell preparation of Enterococcus faecalis strain EC-12 (EC-12) to weaning pigs was previously demonstrated to decrease animal mortality in a STEC-contaminated farm at 0.05% (w/w) dose level. In this study, pigs experimentally infected with STEC were used as a model for ED to evaluate the low dose level of EC-12 to prevent ED. Fifteen 21-day-old pigs were divided into 5 groups: STEC challenge with the basal diet, STEC challenge with EC-12 supplemented at 0.005, 0.01, or 0.05% (w/w) to the basal diet, and no STEC challenge with the basal diet. The challenge was carried out when the animals were 25, 26, and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 32 days of age. The daily weight gain, feed conversion ratio, and palpebral edema were improved by supplementation with 0.05% EC-12, but not by the low dose levels. Accordingly, 0.05% level of supplementation was needed for EC-12 to improve clinical symptoms in weaning piglets infected by STEC.

Improved porcine model for Shiga toxin-producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets.

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 1011  colony-forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum-deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.

The effect of colostrum ingestion during the first 24 hours of life on early postnatal development of piglet immune systems.

It has been suggested that colostrum is important not only for direct protection from pathogens but also for proper development of immune systems in piglets. In this study, we focused on the effect of colostrum ingestion during the first 24 h of life on early postnatal development of piglet immune systems. Thirty-six piglets from five litters were divided into colostrum-fed (CoF) and colostrum-deprived (CoD) groups. The former group was allowed to suckle normally while formula milk was fed to the latter group during the first 24 h of life. At the weaning period, the concentrations of fecal immunoglobulin (Ig) A and plasma IgG as well as the number of blood leukocyte subsets were analyzed. Fecal IgA and plasma IgG concentrations in the CoF group were more than twice as high as those in the CoD group (P < 0.01). In addition, the number of blood B cells was significantly higher in the CoF group than that in the CoD group (P < 0.05). This study demonstrates that colostrum ingestion during the first 24 h plays a significant role in early postnatal development of both mucosal and systemic immunity of piglets.

Comparison of gene expression profiles of T cells in porcine colostrum and peripheral blood.

OBJECTIVE To compare gene expression patterns of T cells in porcine colostrum and peripheral blood. ANIMALS 10 multiparous sows. PROCEDURES Cytotoxic and CD4-CD8 double-positive T cells were separated from porcine colostrum and peripheral blood. Total RNA was extracted. The cDNA prepared from RNA was amplified, labeled, fragmented, and competitively hybridized to DNA microarray slides. The DNA microarray data were validated by use of a real-time reverse-transcription PCR assay, and expression of the genes FOS, NFKBI, IFNG, CXCR6, CCR5, ITGB2, CCR7, and SELL was assessed. Finally, DNA microarray data were validated at the protein level by use of flow cytometry via expression of c-Fos and integrin ß-2. RESULTS Evaluation of gene expression profiles indicated that in contrast to results for peripheral blood, numerous cell-signaling pathways might be activated in colostrum. Profile analysis also revealed that FOS and NFKBI (genes of transcription factors) were involved in most cell-signaling pathways and that expression of these genes was significantly higher in colostral T cells than in peripheral blood T cells. Furthermore, CCR7 and SELL (genes of T-cell differentiation markers) in colostral T cells had expression patterns extremely similar to those found in effector or effector memory T cells. CONCLUSIONS AND CLINICAL RELEVANCE All or most of the T cells in colostrum had an effector-like phenotype and thus were more activated than those in peripheral blood. This gene expression profile would enable T cells to migrate to mammary glands, be secreted in colostrum, and likely contribute to passive immunity provided by sows to newborn pigs.

Supplementation with tetrahydrobiopterin prevents the cardiovascular effects of angiotensin II-induced oxidative and nitrosative stress.

OBJECTIVE: The pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of endothelial nitric oxide synthase (eNOS) activity. When BH4 availability is limited, eNOS does not produce nitric oxide (NO) but instead generates superoxide. BH4 may reverse endothelial dysfunction due to cardiovascular disease, including atherosclerosis, coronary artery disease and hypertension. In this study, the influence of BH4 on cardiovascular parameters and the production of free radicals following angiotensin II (Ang II) infusion was assessed. METHODS: BH4 (20 mg/kg per day in drinking water) was administered with Ang II (300 ng/kg per min subcutaneously, osmotic pump) for 7 days in Sprague-Dawley rats. In addition, BH4 was also given in vehicle-infused rats. RESULTS: Treatment with BH4 significantly prevented some of the effects of Ang II, such as impaired vascular responses to acetylcholine, hypertension and increases in heart weight index values. Treatment with BH4 also significantly reduced Ang II-induced increases in inducible NO synthase expression, nitrotyrosine immunostaining, NO production and superoxide anion formation in rats. CONCLUSION: These results indicate that BH4 might prevent the development of hypertension and myocardial hypertrophy, as well as the Ang II-induced production of superoxide and NO, thereby reducing the production of peroxynitrite. Therefore, BH4 may protect against the cardiovascular manifestations of oxidative and nitrosative stress in this experimental model of Ang II-mediated hypertension.

HMG-CoA reductase inhibitor increases GTP cyclohydrolase I mRNA and tetrahydrobiopterin in vascular endothelial cells.

OBJECTIVE: Endothelial nitric oxide synthase (eNOS) activity is supported by tetrahydrobiopterin (BH4), which appears to be important for generating protective NO but decreases uncoupling formation of superoxide. We investigated the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, in terms of BH4 metabolism in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: We measured the mRNA levels of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the first step of de novo BH4 synthesis, by real-time polymerase chain reaction. The mRNA of GTPCH, as well as of eNOS, was upregulated in HUVECs treated with cerivastatin. This increase was time and dose dependent. Fluvastatin was also observed to enhance GTPCH and eNOS mRNA levels. In parallel with this observation, cerivastatin increased intracellular BH4. Incubating HUVECs with tumor necrosis factor (TNF-alpha) was observed to increase GTPCH mRNA while decreasing eNOS mRNA. In the presence of cerivastatin, the TNF-alpha-mediated increase in GTPCH mRNA was enhanced, and the TNF-alpha-mediated decrease in eNOS mRNA was attenuated. Cerivastatin increased the stability of eNOS mRNA. However, it did not alter the stability of GTPCH mRNA but increased GTPCH gene transcription, as shown by nuclear run-on assays. Preteatment of HUVECs with the selective GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, caused a decrease in intracellular BH4 and decreased citrulline formation after stimulation with ionomycin. Furthermore, the potentiating effect of cerivastatin was decreased by limiting the cellular availability of BH4. CONCLUSIONS: Our data demonstrate that statins elevate GTPCH mRNA, thereby increasing BH4 levels in vascular endothelial cells. In addition to augmenting eNOS expression, statins potentiate GTPCH gene expression and BH4 synthesis, thereby increasing NO production and preventing relative shortages of BH4.

Experimental infection of enterotoxemic Escherichia coli associated with porcine edema disease and its pathologic characteristics in the intestine.

Edema disease (ED) has become frequent in Japan, but no effective method for experimental infection has been developed. We report here the use of a capsule that resistant against gastric digestion to induce the ED in piglets. Four 21-day-old piglets were used. Shiga toxin 2e-producing Escherichia coli (STEC) cell pellet was encapsulated and administered orally. Two pigs received 1.0x10(10) CFU for two days, and the others received 3.9x10(8) CFU for three days. The high-dose group caused the typical clinical ED signs (palpebral edema or neurologic impairment). Eosinophil infiltration, swollen lymphoid follicles, and edema were observed in the ileum. The kidney had the thrombus in the glomerulus.

Statin enhances cytokine-mediated induction of nitric oxide synthesis in vascular smooth muscle cells.

OBJECTIVE: We investigated the effects of the statins, cerivastatin and fluvastatin, on the induction of nitric oxide (NO) production in vascular smooth muscle cells (VSMC) stimulated by interleukin-1beta (IL-1) or in combination with interferon-gamma (IFN). METHODS: We measured NO release, inducible NO synthase (iNOS) mRNA and protein levels, iNOS gene transcription rates, and iNOS mRNA stabilities in cytokine-activated VSMC. We also evaluated nuclear factor (NF)-kappaB activity and tetrahydrobiopterin (BH4) synthesis. RESULTS: NO production induced by cytokines was dose-dependently enhanced by both statins. Incubating VSMC with IL-1/IFN stimulated iNOS mRNA and protein expression. Both statins significantly upregulated IL-1/IFN-stimulated iNOS mRNA and protein expression, and enhanced iNOS gene transcription as shown by nuclear run-on assays. However, they did not alter the stability of iNOS mRNA. Both statins slightly modulated IL-1/IFN-induced NF-kappaB activation, which was not associated with their effect on NO production. Cytokines induce the de novo synthesis of BH4 in VSMC. This event is essential for the induction of NO synthesis, which requires transcriptional induction of the genes that encode not only iNOS but also guanosine triphosphate cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in de novo BH4 synthesis. The synthesis of BH4 and GTPCH mRNA induced by IL-1/IFN were enhanced by both statins. Exogenous mevalonate significantly prevented and geranylgeranylpyrophosphate reversed the stimulatory effect of both statins. Furthermore, the geranylgeranyltransferase I inhibitor GGTI-298 significantly increased IL-1/IFN-induced NO production. CONCLUSION: Our data demonstrated that statins enhance immunostimulants-induced NO production by increasing iNOS gene expression at the transcriptional level via an NF-kappaB-independent pathway. The effect of statins on NO production is due at least partly through blocking the biosynthesis of mevalonate, which prevents isoprenoid biosynthesis. In addition to augmenting iNOS expression, statins potentiate GTPCH gene expression and BH4 synthesis, thereby preventing a relative shortage of BH4 which may shift the balance between NOS-catalyzed generation of protective NO and deleterious reactive oxygen species.

Specific expression of an A-kinase anchoring protein subtype, AKAP-150, and specific regulatory mechanism for Na(+),K(+)-ATPase via protein kinase A in the parotid gland among the three major salivary glands of the rat.

We have examined the expression of A-kinase anchoring protein (AKAP) in the three major salivary glands, i.e. the parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG), of the rat to elucidate the functional relevance between saliva secretion and Na(+),K(+)-ATPase regulation by protein kinase A (PKA)-dependent phosphorylation, since an AKAP subtype, AKAP-150, is known to be involved in the regulation of the ATPase in PG. Although AKAP-150 and its mRNA were clearly detected in the PG, they were hardly detectable in either the SMG or SLG. The membrane-bound form of the RII regulatory subunit of PKA, an index for the total amount of AKAP subtypes and therefore of the anchored PKA holoenzyme, was also undetectable in membranes from the SMG and SLG but was found in the PG; though a substantial and comparable amount of Na(+),K(+)-ATPase was present in all of these membrane preparations. Incubation with [gamma-32P]ATP revealed that Na(+),K(+)-ATPase in the PG membranes was quickly phosphorylated upon the addition of cAMP, whereas the ATPases in the membranes from SMG and SLG were not; though they were readily and equally phosphorylated by the exogenously added PKA catalytic subunit. AKAP-150 in the basolateral membranes of PG acinar cells was co-immunoprecipitated with RII by an anti-RII antiserum; and AKAP-150 and Na(+),K(+)-ATPase were immunohistochemically co-localized predominantly on the basolateral membranes, suggesting a possibility that the ATPase might directly interact with the AKAP to form an ATPase/AKAP/PKA complex or associate with the AKAP, such association being mediated via some scaffolding molecule. Expression of AKAP-150 and quick down-regulation of Na(+),K(+)-ATPase by AKAP-anchored PKA in response to cAMP elevation are characteristics specific to PG among the three major salivary glands, suggesting the presence of PG-specific regulatory mechanisms for saliva production/secretion.

A pharmacologic study on the mechanism of action of Kakkon-to: body temperature elevation and phagocytic activation of macrophages in dogs.

OBJECTIVE: The phagocytic activity of macrophages as a novel approach to scientific elucidation of the effects of Chinese medicines was studied through administration of a kampo preparation, by measuring the rise in body temperature, which is thought to stimulate innate defensive functions of organisms and enhance the immune systems. DESIGN: Using dogs as experimental models, a rise in body temperature following administration of Kakkon-to was observed, and the average number and average rate of phagocytosis of macrophages in blood using latex micro-particles was investigated. RESULTS: The body temperature of the treated animals significantly increased 30 minutes after administration (p<0.01), and remained elevated for more than 5 hours. A comparison of body temperatures before and after administration showed significant increases over controls from 1 to 11 hours, p<0.01; and at 12 hours, p<0.05 after administration. The average number and the average rate of phagocytosis were significantly increased 1 (p<0.05) and 2 (p<0.01) hours after administration. The mean number of phagocytized cells significantly increased (p<0.05) at 1 hour after administration compared with that before administration, and the mean phagocytic rate also increased significantly (p<0.01) 2 hours after administration. Increases (p<0.01) in both the rate of phagocytosis and the number of cells phagocytized were found at every measurement point from 2 to 24 hours after administration. Significant increases (p<0.01) were also observed in both the rate of phagocytosis and the number of cells phagocytized 3 hours after administration, when compared with the control group. CONCLUSION: This paper demonstrates that ingestion of Kakkon-to not only increases the body temperature but also enhances the phagocytic activity of macrophages, an in vivo defense mechanism, suggesting that Kakkon-to contributes to the suppression of multiplication of common cold viruses and influenza viruses, which consequently results in improvement of various symptoms during infection with common cold viruses.