The mode of actions of the Adacolumn therapeutic leucocytapheresis in patients with inflammatory bowel disease: a concise review.

Patients with active inflammatory bowel disease (IBD) have elevated and activated myeloid leucocytes which infiltrate the colonic mucosa in vast numbers. Myeloid leucocytes such as the CD14(+) CD16(+) monocytes are major sources of tumour necrosis factor (TNF)-α, and therefore selective granulocyte/monocyte (GM) adsorption (GMA) should promote remission or enhance efficacy of pharmacological therapy. However, studies in IBD have reported both impressive as well as disappointing efficacy outcomes, indicating that patients' demographic factors might determine responders or non-responders to GMA. Nonetheless, this non-drug intervention has an excellent safety profile, and therapeutic GMA is expected to expand. In this review, attempts have been made to compile an update on the mode of actions (MoA) of the Adacolumn GMA. The MoA of GMA appears to be more than adsorption of excess neutrophils and TNF-producing CD14(+) CD16(+) monocytes per se. Adsorbed GMs release interleukin (IL)-1 receptor antagonist, hepatocyte growth factor and soluble TNF receptors, which are anti-inflammatory. Additionally, a sustained increase in lymphocytes including the regulatory CD4(+) CD25(+) T cells (lymphocyte sparing) is seen post-GMA. The impact of GMA on the immune system is potentially very interesting in the context of treating immune-related diseases. Future studies are expected to add intriguing insights to the MoA of GMA.

Desensitization to parathyroid hormone in renal cells from aged rats is associated with alterations in G-protein activity.

Parathyroid hormone (PTH)-stimulated Na+/Ca2+ exchange activity, but not forskolin-sensitive Na+-dependent Ca2+ efflux, was blunted in renal cortical cells from aged rats. PTH-sensitive adenylate cyclase activity in renal membranes from senescent rats also declined, but forskolin-stimulated activity did not change. In addition, cholera toxin- and pertussis toxin-stimulated Na+-dependent Ca2+ efflux and cAMP formation were blunted in cells from aged animals. Further, cells from aged rats had decreased Gs-alpha and Gi-alpha proteins, as detected by ADP-ribosylation. These findings would be consistent with the proposal of an age-associated heterologous desensitization that involved the G-proteins. Serum concentrations of iPTH were increased in the old rat, suggesting that the desensitization to PTH in the aging rat represented an adaptive response to prolonged stimulation by the hormone. This hypothesis was supported by the findings that the attenuated PTH-sensitive Na+/Ca2+ exchange activity, cAMP formation, and adenylate cyclase activity in cells from old rats could be reversed by parathyroidectomy. The decreased label in cholera toxin-catalyzed ADP-ribosylated Gs-alpha and pertussis toxin catalyzed ADP-ribosylated Gi-alpha found in cells from aged rats was also largely negated by the surgery. In conclusion, the results suggest that the age-related blunting in the responses of renal cells to PTH was associated with a deficit in G-protein function and that this alteration could be reversed by removal of the parathyroid gland.

Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene.

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH(2)-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (alpha PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of alpha PIX (DeltaCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated alphaPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted alphaPIX (Delta SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of alphaPIX (Delta CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas alphaPIX (Delta SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the alphaPIX (Delta CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that alphaPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.

alphaPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase.

p21-activated kinase (PAK) is a common effector protein of the small GTPases Cdc42 and Rac, leading to the activation of downstream mitogen activated protein kinases. PAK also mediates polarized cytoskeletal changes induced by these GTPases. The recently identified PAK-interacting exchange factor (PIX) acts as a guanine nucleotide exchange factor on Rac, and colocalizes with PAK in a focal complex, but little is known about the associated signaling cascades, including upstream activators of PIX. In this study, we show that one of the isoforms of PIX, alphaPIX, is activated by signaling cascades from the platelet-derived growth factor (PDGF) receptor and EphB2 receptor, and from integrin-induced signaling through phosphatidylinositol 3-kinase (PI3-kinase). alphaPIX is activated by forming a complex with these receptors either via association with PAK and Nck, or direct association with the p85 regulatory subunit of PI3-kinase. Synthetic phosphoinositide and membrane targeted PI3-kinase augmented the alphaPIX activity in vivo. In Xenopus, aggregates of mesodermal cells derived from embryos microinjected with alphaPIX significantly increased the peripheral spreading on fibronectin substrate in response to PDGF through PI3-kinase. These results indicate that alphaPIX is activated by PI3-kinase, and is involved in the receptor mediated signaling leading to the activation of the kinase activity of PAK, and the migration of mesodermal cells on extracellular matrix.

Effects of aging on the microclimate pH of the rat jejunum.

The acidic microclimate layer in the vicinity of the cell surface of mammalian jejunum is important for absorption of some nutrients, such as small peptides and folate. The present study was undertaken to investigate the effect of aging on the cell surface pH (microclimate pH) of the jejunum of rats. The microclimate pH was measured in vitro in superfused preparations using single-barreled pH-sensitive microelectrodes filled with a liquid ion exchanger. The thickness of the microclimate layer was estimated by reading the distance of microelectrode advancements. The existence of a microclimate pH in the jejunum was confirmed in the senescent rats, but the value of the microclimate pH was significantly higher in the senescent (24 mo) rats (6.52 +/- 0.02) than in the young-adult (6 mo) rats (6.09 +/- 0.01) (P < 0.01). Na+ removal from the perfusate or the addition of amiloride elevated the pH in the senescent rats as well as in the young-adult rats. The microclimate layer was slightly thinner in the senescent rats than in the young-adult rats. The acidity of the microclimate layer of intestinal surface is lower in senescent animals than in the young-adult ones. One of reasons for this is the thinner mucus layer in senescent animals.

Effects of genotypic differences in CYP2C19 status on cure rates for Helicobacter pylori infection by dual therapy with rabeprazole plus amoxicillin.

Rabeprazole is a potent proton pump inhibitor and is mainly reduced to thioether rabeprazole by a non-enzymatic pathway and partially metabolized to demethylated rabeprazole by CYP2C19 in the liver. We intended to determine a cure rate for Helicobacter pylori infection by dual rabeprazole/amoxicillin therapy in relation to CYP2C19 genotype status prospectively. Ninety-seven patients with gastritis and H. pylori infection completed the dual therapy with 10 mg of rabeprazole bid and 500 mg of amoxicillin tid for 2 weeks. At 1 month after treatment, cure of H. pylori infection was assessed on the basis of histology, a rapid urease test, culture, polymerase chain reaction (PCR), and 13C-urea breath test. CYP2C19 genotype status was determined by a PCR-restriction fragment length polymorphism method. Of the 97 patients, 33 were homozygous extensive metabolizers (homEM), 48 were heterozygous extensive metabolizers (hetEM), and 16 were poor metabolizers (PM). Cure of H. pylori infection was achieved in 79 of the 97 patients (81.4%, 95%CI = 71.9-88.7). Significant differences in cure rates among the homEM, hetEM, and PM groups were observed; 60.6% (95%CI = 42.1-77.3), 91.7% (95%CI = 80.0-97.7), and 93.8% (95%CI = 69.8-99.8), respectively (P = 0.0007). Twelve patients without cure after initial treatment (10 homEMs and 2 hetEMs) were successfully retreated with rabeprazole 10 mg q.i.d. and amoxicillin 500 mg q.i.d. for 2 weeks. The cure rates for H. pylori infection by dual rabeprazole/amoxicillin therapy depended on the CYP2C19 genotype status. This dual therapy appears to be effective for hetEM and PM patients. However, high dose dual rabeprazole/amoxicillin therapy was effective even for homEM patients. Therefore, the genotyping test of CYP2C19 appears to be a clinically useful tool for the optimal dual treatment with rabeprazole plus amoxicillin.

Cure of refractory duodenal ulcer and infection caused by Helicobacter pylori by high doses of omeprazole and amoxicillin in a homozygous CYP2C19 extensive metabolizer patient.

A 53-year old female patient with duodenal ulcer and Helicobacter pylori infection was treated three times with a proton pump inhibitor-based triple therapy, such as lansoprazole-clarithromycin-amoxicillin (INN, amoxicilline) and lansoprazole-minocycline-cefaclor. However, the H pylori infection was not cured. A culture test revealed that her infection was a clarithromycin-resistant but amoxicillin-sensitive strain of H pylori. Moreover, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that she was a homozygous extensive metabolizer of cytochrome P450 (CYP) 2C19 (wt/wt). The usual dose of the proton pump inhibitor was therefore assumed to be insufficient for her and then she was treated with a high dose of omeprazole (120 mg/day) and amoxicillin (2,250 mg/day) for 2 weeks. The H pylori infection and the ulcer lesion were then cured. One of the factors associated with success or failure of cure of H pylori infection by the proton pump inhibitor-based triple therapy appeared to be CYP2C19 genotype status. Dual treatment with a sufficient dose of a proton pump inhibitor plus amoxicillin could cure H pylori infection even after the failure to cure H pylori infection by a usual proton pump inhibitor-based triple therapy in patients with the wt/wt homozygous extensive metabolizer genotype of CYP2C19.

Effects of clarithromycin on the metabolism of omeprazole in relation to CYP2C19 genotype status in humans.

BACKGROUND AND PURPOSE: A triple therapy with omeprazole, amoxicillin (INN, amoxicilline), and clarithromycin is widely used for the eradication of Helicobacter pylori. Omeprazole and clarithromycin are metabolized by CYP2C19 and CYP3A4. This study aimed to elucidate whether clarithromycin affects the metabolism of omeprazole. METHODS: After administration of placebo or 400 mg clarithromycin twice a day for 3 days, 20 mg omeprazole and placebo or 400 mg clarithromycin were administered to 21 healthy volunteers. Plasma concentrations of omeprazole and clarithromycin and their metabolites were determined before and 1, 2, 3, 5, 7, 10, and 24 hours after dosing. CYP2C19 genotype status was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Subjects were classified into three groups on the basis of PCR-RFLP analyses for CYP2C19: homozygous extensive metabolizer group (n = 6), heterozygous extensive metabolizer group (n = 11), and poor metabolizer group (n = 4). Mean area under the plasma concentration-time curves from 0 to 24 hours (AUC) of omeprazole in the homozygous extensive metabolizer, heterozygous extensive metabolizer, and poor metabolizer groups were significantly increased by clarithromycin from 383.9 to 813.1, from 1001.9 to 2110.4, and from 5589.7 to 13098.6 ng x h/mL, respectively. There were significant differences in the mean AUC values of clarithromycin among the three groups. CONCLUSION: Clarithromycin inhibits the metabolism of omeprazole. Drug interaction between clarithromycin and omeprazole may underlie high eradication rates achieved by triple therapy with omeprazole, amoxicillin, and clarithromycin.

CYP2C19 genotype status and effect of omeprazole on intragastric pH in humans.

OBJECTIVE: Omeprazole is metabolized by genetically determined S-mephenytoin 4'-hydroxylase (CYP2C19) in the liver. This study aimed to determine whether the effect of omeprazole on intragastric pH depends on CYP2C19 genotype status. METHODS: CYP2C19 genotype status for 2 mutations associated with the poor metabolizer phenotype was determined by a polymerase chain reaction-restriction fragment length polymorphism method in 16 healthy volunteers. Helicobacterpylori status was determined by serology and the [13C]urea breath test. After a single oral administration of 20 mg omeprazole or a placebo, intragastric pH values were recorded for 24 hours. Plasma levels of omeprazole and its 2 metabolites and gastrin were measured before and 1, 2, 3, 5, 7, 10, and 24 hours after administration. RESULTS: Fifteen of the 16 subjects were H pylori negative. Five of the 15 subjects were homozygous extensive metabolizers, 4 were heterozygous extensive metabolizers, and 6 were poor metabolizers. After omeprazole administration, significant differences in mean intragastric pH values and plasma levels of gastrin, omeprazole and its metabolites were observed among the 3 groups, whereas no significant differences in these parameters were observed with the placebo administration. CONCLUSIONS: The effect of omeprazole on intragastric pH significantly depends on CYP2C19 genotype status. The genotyping test of CYP2C19 may be useful for an optimal prescription of omeprazole.

Effect of genotypic differences in CYP2C19 on cure rates for Helicobacter pylori infection by triple therapy with a proton pump inhibitor, amoxicillin, and clarithromycin.

BACKGROUND: Proton pump inhibitors such as omeprazole and lansoprazole are mainly metabolized by CYP2C19 in the liver. The therapeutic effects of proton pump inhibitors are assumed to depend on CYP2C19 genotype status. OBJECTIVE: We investigated whether CYP2C19 genotype status was related to eradication rates of H pylori by triple proton pump inhibitor-clarithromycin-amoxicillin (INN, amoxicilline) therapy and attempted to establish a strategy for treatment after failure to eradicate H pylori. METHODS: A total of 261 patients infected with H pylori completed initial treatment with 20 mg of omeprazole or 30 mg of lansoprazole twice a day, 200 mg of clarithromycin three times a day, and 500 mg of amoxicillin three times a day for 1 week. CYP2C19 genotypes of patients were determined with polymerase chain reaction-restriction fragment length polymorphism analysis. Patients without eradication after initial treatment were retreated with 30 mg of lansoprazole four times daily and 500 mg of amoxicillin four times daily for 2 weeks. RESULTS: Eradication rates for H pylori were 72.7% (95% confidence interval, 64.4%-81.8%), 92.1% (confidence interval, 86.4%-97.3%), and 97.8% (confidence interval, 88.5%-99.9%) in the homozygous extensive, heterozygous extensive, and poor metabolizer groups, respectively. Thirty-four of 35 patients without eradication had an extensive metabolizer genotype of CYP2C19. Nineteen of those patients were infected with clarithromycin-resistant strains of H pylori. However, there were no amoxicillin-resistant strains of H pylori. Re-treatment of H pylori infection with dual high-dose lansoprazole-amoxicillin therapy succeeded in 30 of 31 patients with extensive metabolizer genotype of CYP2C19. CONCLUSION: The majority of patients without initial eradication of H pylori had an extensive metabolizer CYP2C19 genotype but were successfully re-treated with high doses of lansoprazole and an antibiotic to which H pylori was sensitive, such as amoxicillin, even when the patients were infected with clarithromycin-resistant strains of H pylori.

Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro.

An in vitro assay system to detect tyrosylprotein sulfotransferase (TPST) activity of higher plant cells was established, using synthetic oligopeptides based on the deduced amino acid sequence of a phytosulfokine-alpha (PSK-alpha) precursor. TPST activity was found in microsomal membrane fractions of rice, asparagus and carrot cells and it was confirmed that acidic amino acid residues adjacent to the tyrosine residues of acceptor peptides were essential to the sulfation reaction. The asparagus TPST exhibited a broad pH optimum of 7.0-8.5, required manganese ions for maximal activity and appeared to be a membrane-bound protein localized in the Golgi apparatus. These enzymes should be defined as a new class of plant sulfotransferases that catalyze tyrosine O-sulfation of a PSK-alpha precursor and other unknown proteins.

Permissive role of L-thyroxine in induction of pancreatic amylase by cortisol in neonatal rats.

The effect of L-thyroxine (T4) on amylase activity in the developing rat pancreas has been investigated. Administration of T4 (0.2 microgram/g body wt) alone to intact rats on days 5-10 after birth did not induce pancreatic amylase but the enzyme was induced significantly by daily injection of cortisol (10 microgram/g body wt) alone into intact rats over the same period. In thyroidectomized, adrenalectomized rats pancreatic amylase was not induced by the injection of cortisol alone but it was induced by the administration of cortisol plus T4. Increase in enzyme activity was much less in thyroidectomized animals than in intact animals. These results suggested that T4 does not have a direct effect in increasing pancreatic amylase activity but plays a permissive role in increasing enzyme activity.

Regulation of renal sodium calcium exchange by PTH: alteration with age.

Parathyroid hormone, when incubated with renal cells acting in vivo and in vitro, increased Na+/Ca2+ exchange activity. The effect of parathyroid hormone was specific for biologically active analogs and could be mimicked by cAMP and forskolin. Parathyroid hormone-sensitive Na+/Ca2+ exchange activity was markedly blunted in cells from senescent rats. Parathyroid hormone-stimulated adenylate cyclase was also decreased in aging. In contrast, forskolin-stimulated Na(+)-dependent Ca2+ efflux and adenylate cyclase did not change with senescence. Decrease of PTH binding sites was observed in cells from old rats. Further, cells from 24-month-old rats had decreased Gs and Gi proteins, as detected by ADP-ribosylation. Since serum iPTH level was elevated in the old rat and could contribute to the desensitization to PTH, we tested this hypothesis by comparing sham-operated and PTX animals. The decreases in PTH-sensitive Na+/Ca2+ exchange activity and adenylate cyclase activity in cells from 24-month-old rats could be completely negated by parathyroidectomy. Decrease in PTH binding sites and contents of Gs and Gi in cells from aged-rats was partially negated by the surgery. In conclusion, our results suggested that the age related blunting in responses of renal cells to PTH was due, at least in part, to the elevated serum iPTH level in old rats.

Effect of Helicobacter pylori infection and its eradication on nutrition.

AIMS: To investigate the effects of Helicobacter pylori infection and eradication on nutrition. METHODS: The body weight, height, blood pressure, gastric juice pH and fasting serum levels of glucose, total protein, albumin, total cholesterol and triglyceride were measured in H. pylori-positive and H. pylori-negative subjects, and the effect of eradication of H. pylori on these parameters was determined. The development of gastro-oesophageal reflux disease after treatment was also examined. Eight patients underwent a pancreatic function test before and after H. pylori eradication therapy. RESULTS: The incidence of hypoproteinaemia in H. pylori-positive subjects was significantly higher than that in H. pylori-negative subjects. After eradication of H. pylori, the gastric juice pH values were significantly decreased, and the body weight and serum levels of total cholesterol, total protein and albumin were significantly increased. The incidence of hyperlipidaemia significantly increased and that of hypoproteinaemia significantly decreased in the group with eradication. Pancreatic function improved significantly after eradication of H. pylori. No significant changes in these parameters were observed in the group without eradication. Obese patients had a higher risk of the development of gastro-oesophageal reflux disease after eradication of H. pylori infection. CONCLUSIONS: The eradication of H. pylori appears to improve some nutritional parameters.

Involvement of cyclooxygenase-2 in hyperplastic gastritis induced by Helicobacter pylori infection in C57BL/6 mice.

BACKGROUND AND AIMS: The hyperplastic changes observed in Helicobacter pylori-associated gastritis have been considered to increase the risk of gastric cancer. The aim of this study was to determine whether cyclooxygenase-2 is involved in the hyperplastic changes in mice infected with H. pylori. METHODS: Seven-week-old, male C57BL/6 mice (n=40) were inoculated with the Sydney strain of H. pylori. Control mice (n=40) were treated with vehicle only. Half of the infected and control mice were fed an experimental diet containing etodolac (10 mg/kg/day) from 1 week after inoculation until the end of the experiment. The thickness of gastric pits, COX-2 mRNA and protein levels, and prostaglandin E2 (PGE2) levels in the gastric mucosa were determined before and 12, and 24 weeks after inoculation. RESULTS: The thickness of gastric pits, COX-2 mRNA and protein levels, and PGE2 levels were significantly increased at 24 weeks after inoculation of H. pylori compared with the control groups. Treatment with etodolac resulted in significant decreases in PGE2 production and in the thickness of gastric pits in the infected groups at 24 weeks after inoculation. CONCLUSIONS: Our findings suggest that COX-2 is involved in the development of hyperplastic gastritis caused by H. pylori infection via the production of PGE2.

Comparison of lansoprazole and famotidine for gastric acid inhibition during the daytime and night-time in different CYP2C19 genotype groups.

BACKGROUND: The acid inhibitory effect of lansoprazole depends on the S-mephenytoin 4'-hydroxylase (CYP2C19) genotype status. The effect of famotidine is independent of this genotype. AIM: To investigate the acid inhibitory effects of lansoprazole vs. famotidine during the daytime and night-time with reference to different CYP2C19 genotypes. METHODS: Fifteen healthy volunteers were given 20 mg famotidine twice a day or 30 mg lansoprazole once a day for 8 days. On post-dose day 8, 24-h intragastric pH monitoring was performed. RESULTS: During the daytime, the intragastric pH with lansoprazole was significantly higher than that with famotidine in the heterozygous extensive metabolizer group, whereas no significant difference was observed in the homozygous extensive metabolizer group. During the night-time, the intragastric pH with famotidine was quite similar to that with lansoprazole in the heterozygous extensive metabolizer and poor metabolizer groups. However, during the night-time, the intragastric pH with famotidine was significantly higher than that with lansoprazole in the homozygous extensive metabolizer group. CONCLUSIONS: An insufficient acid inhibition by lansoprazole during the night-time in the homozygous extensive metabolizer group could be compensated for by famotidine. CYP2C19 genotype testing appears to be useful for predicting the optimal acid inhibitory drug treatment collated with circadian intragastric pH change.

Effects of CYP2C19 genotypic differences in the metabolism of omeprazole and rabeprazole on intragastric pH.

BACKGROUND: Omeprazole is mainly metabolized in the liver by CYP2C19, a genetically determined enzyme, whereas rabeprazole is mainly reduced non-enzymatically and partially metabolized by CYP2C19. The therapeutic effects of rabeprazole are therefore assumed to be less affected by an individual's CYP2C19 status. AIM: To investigate the acid inhibitory effects and plasma levels of omeprazole and rabeprazole with reference to different CYP2C19 genotypes. METHODS: Fifteen healthy volunteers took a daily dose of 20 mg of omeprazole or rabeprazole for 8 days. On post-dose days 1 and 8, 24-h profiles of intragastric pH were recorded and plasma concentrations of omeprazole, rabeprazole and their metabolites were determined. RESULTS: After single and repeated doses of omeprazole, the intragastric pH values and plasma concentrations of omeprazole and its metabolites were significantly dependent on the CYP2C19 genotype. Significant differences in the same kinetic and dynamic parameters were also observed after single doses of rabeprazole. Although the plasma levels of rabeprazole differed among the different CYP2C19 genotype groups after repeated doses, no significant differences in intragastric pH values were observed. CONCLUSIONS: The acid inhibitory effects of omeprazole and rabeprazole are significantly dependent on the CYP2C19 genotype status, as well as on their intrinsic pharmacokinetic and pharmacodynamic characteristics and dosing schemes.

Effects of aging on the regulation of intracellular pH in the rat jejunum.

Jejunal villus cells from young-adult (6 months) and senescent (24 months) male Wistar rats were studied to evaluate the effect of aging on intracellular pH (pHi) regulation. pHi was measured by quantitative fluorescence microscopy by using BCECF-AM [2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein acetoxy methylester] under basal conditions and after inducing cytoplasmic acidification with pulsed NH4Cl. In the senescent rats, the recovery rate from the acidified levels was significantly lower than that in the young-adult rats (.208 +/- .005 vs .255 +/- .004 pH units/min). The relationship between pHi recovery and external Na+ concentration followed Michaelis-Menten type kinetics, the maximum velocity (Vmax) of alkalinization being significantly lower in the senescent rats than in the young-adult rats (.227 +/- .033 vs .297 +/- .024 pH units/min). These results indicate that the recovery of pHi from an acidic level was slower in the senescent rats, due to the reduced activity of Na+/H+ exchange as revealed by the decreased Vmax value.

Synergistic activation of brain adenylate cyclase by calmodulin, and either GTP or catecholamines including dopamine.

The effects of calmodulin (CaM), guanosine triphosphate (GTP) and dopaminergic or beta-adrenergic agonists on the activities of adenylate cyclase were studied in EGTA-washed lysed synaptosomal membranes from rat striatum and cerebral cortex. Based on the free calcium ion concentration-dependence of the enzymic activity, it was found that the stimulatory effect of CaM and GTP on adenylate cyclase was synergistic with maximum activation at pCa 6.2 for both striatal and cortical membranes. This was not due to the effect of CaM-dependent phosphodiesterase because exogenous CaM did not affect particulate phosphodiesterase activity. Added CaM not only enhanced the adenylate cyclase activity but acted cooperatively with dopaminergic or beta-adrenergic agonists in the presence of GTP. Most marked was enhancement found for the striatal SKF 38393- (a specific D1 agonist) and the cortical isoproterenol-dependent activities. A synergism was also found for CaM and forskolin. These findings strongly suggest that CaM is involved in the striatal dopaminergic as well as in the cortical beta-adrenergic systems.