Construction of chromosome markers from the Lake Victoria cichlid Paralabidochromis chilotes and their application to comparative mapping.

Cichlid fishes in the African Great Lakes are known as a spectacular example of adaptive radiation in vertebrates. Four linkage maps have been constructed to identify the genes responsible for adaptation and speciation, and the genetic linkages of those genes are assumed to play an important role during adaptive evolution. However, it is difficult to analyze such linkages because the linkage groups of one species do not match well with those of the other species. Chromosome markers are a powerful tool for the direct identification of linkage homology between different species. We used information about the linkage map of the Lake Malawi cichlid (Labeotropheus fuelleborni/Metriaclima zebra) to isolate bacterial artificial chromosome (BAC) clones from the BAC library of Paralabidochromis chilotes, Lake Victoria. We identified 18 of 22 P. chilotes chromosomes by single- and multi-color BAC fluorescence in situ hybridization using 19 BAC clones. Comparative mapping with the chromosome markers of P. chilotes in Astatotilapia burtoni (2n = 40) from Lake Tanganyika revealed the chromosome rearrangements that have occurred in this lineage. These chromosome markers will be useful for delineating the process of genome and chromosome evolution in African species.

Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases.

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 domain rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here, we report the atomic models of 18 catalytic intermediates of the V1 domain of V/A-ATPase under different reaction conditions, determined by single particle cryo-EM. The models reveal that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

Strenuous swimming raises blood non-enzymatic antioxidant capacity in rats.

The non-enzymatic antioxidant system protects blood components from oxidative damage and/or injury. Herein, plasma non-enzymatic antioxidant capacity after acute strenuous swimming exercise (Exe) and exercise until exhaustion (Exh) was measured in rats. The experiments were carried out in never exposed (Nex) and pre-exposed (Pex) groups. The Nex group did not undergo any previous training before the acute strenuous swimming test and the Pex group was submitted to daily swimming for 10 min in the first week and 15 min per day in the second week before testing. Plasma glucose, lactate, and pyruvate were measured and plasma total protein sulfhydryl groups (thiol), trolox equivalent antioxidant capacity (TEAC), ferric reducing ability of plasma (FRAP), and total radical-trapping antioxidant parameter (TRAP) levels were evaluated. There were marked increases in plasma lactate concentrations (Nex-Control 1.31±0.20 vs NexExe 4.16±0.39 vs NexExh 7.19±0.67) and in thiol (Nex-Control 271.9±5.6 vs NexExh 314.7±5.7), TEAC (Nex-Control 786.4±60.2 vs NexExh 1027.7±58.2), FRAP (Nex-Control 309.2±17.7 vs NexExh 413.4±24.3), and TRAP (Nex-Control 0.50±0.15 vs NexExh 2.6±0.32) levels after acute swimming and/or exhaustion. Also, there were increased plasma lactate concentrations (Pex-Control 1.39±0.15 vs PexExe 5.22±0.91 vs PexExh 10.07±0.49), thiol (Pex-Control 252.9±8.2 vs PexExh 284.6±6.7), FRAP (Pex-Control 296.5±15.4 vs PexExh 445.7±45.6), and TRAP (Pex-Control 1.8±0.1 vs PexExh 4.6±0.2) levels after acute swimming and/or exhaustion. Lactate showed the highest percent of elevation in the Nex and Pex groups. In conclusion, plasma lactate may contribute to plasma antioxidant defenses, and the TRAP assay is the most sensitive assay for assessing plasma non-antioxidant capacity after strenuous exercise.

A Japanese Multicenter Study on PET and Other Biomarkers for Subjects with Potential Preclinical and Prodromal Alzheimer's Disease.

BACKGROUND: PET (positron emission tomography) and CSF (cerebrospinal fluid) provide the "ATN" (Amyloid, Tau, Neurodegeneration) classification and play an essential role in early and differential diagnosis of Alzheimer's disease (AD). OBJECTIVE: Biomarkers were evaluated in a Japanese multicenter study on cognitively unimpaired subjects (CU) and early (E) and late (L) mild cognitive impairment (MCI) patients. MEASUREMENTS: A total of 38 (26 CU, 7 EMCI, 5 LMCI) subjects with the age of 65-84 were enrolled. Amyloid-PET and FDG-PET as well as structural MRI were acquired on all of them, with an additional tau-PET with 18F-flortaucipir on 15 and CSF measurement of Aß1-42, P-tau, and T-tau on 18 subjects. Positivity of amyloid and tau was determined based on the positive result of either PET or CSF. RESULTS: The amyloid positivity was 13/38, with discordance between PET and CSF in 6/18. Cortical tau deposition quantified with PET was significantly correlated with CSF P-tau, in spite of discordance in the binary positivity between visual PET interpretation and CSF P-tau in 5/8 (PET-/CSF+). Tau was positive in 7/9 amyloid positive and 8/16 amyloid negative subjects who underwent tau measurement, respectively. Overall, a large number of subjects presented quantitative measures and/or visual read that are close to the borderline of binary positivity, which caused, at least partly, the discordance between PET and CSF in amyloid and/or tau. Nine subjects presented either tau or FDG-PET positive while amyloid was negative, suggesting the possibility of non-AD disorders. CONCLUSION: Positivity rate of amyloid and tau, together with their relationship, was consistent with previous reports. Multicenter study on subjects with very mild or no cognitive impairment may need refining the positivity criteria and cutoff level as well as strict quality control of the measurements.

Chlorophyll treatment combined with photostimulation increases glycolysis and decreases oxidative stress in the liver of type 1 diabetic rats.

Photodynamic therapy (PDT) promotes cell death, and it has been successfully employed as a treatment resource for neuropathic complications of diabetes mellitus (T1DM) and hepatocellular carcinoma. The liver is the major organ involved in the regulation of energy homeostasis, and in pathological conditions such as T1DM, changes in liver metabolic pathways result in hyperglycemia, which is associated with multiple organic dysfunctions. In this context, it has been suggested that chlorophyll-a and its derivatives have anti-diabetic actions, such as reducing hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, but these effects have not yet been proven. Thus, the biological action of PDT with chlorophyll-a on hepatic parameters related to energy metabolism and oxidative stress in T1DM Wistar rats was investigated. Evaluation of the acute effects of this pigment was performed by incubation of isolated hepatocytes with chlorophyll-a and the chronic effects were evaluated by oral treatment with chlorophyll-based extract, with post-analysis of the intact liver by in situ perfusion. In both experimental protocols, chlorophyll-a decreased hepatic glucose release and glycogenolysis rate and stimulated the glycolytic pathway in DM/PDT. In addition, there was a reduction in hepatic oxidative stress, noticeable by decreased lipoperoxidation, reactive oxygen species, and carbonylated proteins in livers of chlorophyll-treated T1DM rats. These are indicators of the potential capacity of chlorophyll-a in improving the status of the diabetic liver.

Effects of seed- and pollen-mediated gene dispersal on genetic structure among Quercus salicina saplings.

We evaluated the effects of seed- and pollen-mediated gene dispersal on genetic structure among Quercus salicina saplings. Parentage analysis using 10 microsatellite markers indicated that the 111 adult trees located within a 11.56 ha plot in the Tatera Forest Reserve, Japan, included only one parent of 44.2% and both parents of 40.7% of the 226 saplings located in a 1-ha core plot at its center. Coancestry (F(ij)) estimates indicated that there was strong genetic structure among the saplings. The numbers of pairs of full- and half-siblings were high among neighboring saplings, suggesting that there was strong maternal half-sibling family structure among the saplings around their seed parents, probably generated by the spatially limited seed dispersal and the small extent of overlapping seed shadows owing to the low density of adults. The frequencies also suggest that the maternal half-sibling families are interspersed with full-siblings, produced by correlated mating, probably because pollination frequency depends on the distance between parents. The frequencies of pairs of half-siblings decreased as the distance between saplings increased, but did not fall to zero even at distances up to the 90-95 m class, suggesting that paternal half-siblings originating from correlated paternity were widely distributed owing to extensive pollen flow. We separately examined the genetic structure for maternal and paternal alleles in the saplings. Unsurprisingly, very strong genetic structure was detected for maternal alleles. However, weak (but significant) genetic structure was also detected for paternal alleles. Therefore, pollen dispersal may affect the extent of genetic structure as well as seed dispersal.

Asymmetric alloy formation at the Fe-on-Ti and Ti-on-Fe interfaces.

The Fe-on-Ti and Ti-on-Fe interfaces were studied experimentally by Mössbauer spectroscopy (MS), transmission electron microscopy (TEM) and x-ray reflectometry (XRR) on Ti/Fe/Ti trilayers grown on Si(1 1 1) substrates by vacuum evaporation. The nanoscale structure and composition were explored in cross sections using TEM, the layer structure and the interface widths by specular x-ray reflectometry. MS was applied to identify the interface alloy phases and to determine the pure and alloyed Fe layer fractions. The experimental results were compared with molecular dynamics (MD) simulations of layer growth on Fe or Ti underlayers of different orientations. The concentration distributions provided by MD simulations show an asymmetry at the interfaces in the layer growth direction. The transition is atomically sharp at the Ti-on-Fe interface for the (0 0 1) and (1 1 0) crystallographic orientations of the Fe underlayer, while it spreads over a few atomic layers for Fe(1 1 1) underlayer and for all studied Ti underlayer orientations at the Fe-on-Ti interface. MS and XRR data on Ti/Fe/Ti trilayers confirm the asymmetry between the bottom and top Fe interface, but the inferred interface widths considerable exceed those deduced from the MD simulations.

Strenuous swimming raises blood non-enzymatic antioxidant capacity in rats

The non-enzymatic antioxidant system protects blood components from oxidative damage and/or injury. Herein, plasma non-enzymatic antioxidant capacity after acute strenuous swimming exercise (Exe) and exercise until exhaustion (Exh) was measured in rats. The experiments were carried out in never exposed (Nex) and pre-exposed (Pex) groups. The Nex group did not undergo any previous training before the acute strenuous swimming test and the Pex group was submitted to daily swimming for 10 min in the first week and 15 min per day in the second week before testing. Plasma glucose, lactate, and pyruvate were measured and plasma total protein sulfhydryl groups (thiol), trolox equivalent antioxidant capacity (TEAC), ferric reducing ability of plasma (FRAP), and total radical-trapping antioxidant parameter (TRAP) levels were evaluated. There were marked increases in plasma lactate concentrations (Nex-Control 1.31±0.20 vs NexExe 4.16±0.39 vs NexExh 7.19±0.67) and in thiol (Nex-Control 271.9±5.6 vs NexExh 314.7±5.7), TEAC (Nex-Control 786.4±60.2 vs NexExh 1027.7±58.2), FRAP (Nex-Control 309.2±17.7 vs NexExh 413.4±24.3), and TRAP (Nex-Control 0.50±0.15 vs NexExh 2.6±0.32) levels after acute swimming and/or exhaustion. Also, there were increased plasma lactate concentrations (Pex-Control 1.39±0.15 vs PexExe 5.22±0.91 vs PexExh 10.07±0.49), thiol (Pex-Control 252.9±8.2 vs PexExh 284.6±6.7), FRAP (Pex-Control 296.5±15.4 vs PexExh 445.7±45.6), and TRAP (Pex-Control 1.8±0.1 vs PexExh 4.6±0.2) levels after acute swimming and/or exhaustion. Lactate showed the highest percent of elevation in the Nex and Pex groups. In conclusion, plasma lactate may contribute to plasma antioxidant defenses, and the TRAP assay is the most sensitive assay for assessing plasma non-antioxidant capacity after strenuous exercise.

Strenuous swimming raises blood non-enzymatic antioxidant capacity in rats

The non-enzymatic antioxidant system protects blood components from oxidative damage and/or injury. Herein, plasma non-enzymatic antioxidant capacity after acute strenuous swimming exercise (Exe) and exercise until exhaustion (Exh) was measured in rats. The experiments were carried out in never exposed (Nex) and pre-exposed (Pex) groups. The Nex group did not undergo any previous training before the acute strenuous swimming test and the Pex group was submitted to daily swimming for 10 min in the first week and 15 min per day in the second week before testing. Plasma glucose, lactate, and pyruvate were measured and plasma total protein sulfhydryl groups (thiol), trolox equivalent antioxidant capacity (TEAC), ferric reducing ability of plasma (FRAP), and total radical-trapping antioxidant parameter (TRAP) levels were evaluated. There were marked increases in plasma lactate concentrations (Nex-Control 1.31±0.20 vs NexExe 4.16±0.39 vs NexExh 7.19±0.67) and in thiol (Nex-Control 271.9±5.6 vs NexExh 314.7±5.7), TEAC (Nex-Control 786.4±60.2 vs NexExh 1027.7±58.2), FRAP (Nex-Control 309.2±17.7 vs NexExh 413.4±24.3), and TRAP (Nex-Control 0.50±0.15 vs NexExh 2.6±0.32) levels after acute swimming and/or exhaustion. Also, there were increased plasma lactate concentrations (Pex-Control 1.39±0.15 vs PexExe 5.22±0.91 vs PexExh 10.07±0.49), thiol (Pex-Control 252.9±8.2 vs PexExh 284.6±6.7), FRAP (Pex-Control 296.5±15.4 vs PexExh 445.7±45.6), and TRAP (Pex-Control 1.8±0.1 vs PexExh 4.6±0.2) levels after acute swimming and/or exhaustion. Lactate showed the highest percent of elevation in the Nex and Pex groups. In conclusion, plasma lactate may contribute to plasma antioxidant defenses, and the TRAP assay is the most sensitive assay for assessing plasma non-antioxidant capacity after strenuous exercise.

In vitro activation of bovine adrenal tyrosine hydroxylase by rabbit skeletal muscle actin: evidence for a possible role of cytoskeletal elements as an activator for cytoplasmic enzymes.

Tyrosine hydroxylase prepared from the soluble fraction of bovine adrenal medulla was markedly activated by rabbit skeletal muscle G-actin, and this activation was accompanied by a decrease in the apparent Km of the enzyme for the pterin cofactor. The activating effect of G-actin on the soluble enzyme was still observed in the medium containing a high concentration of salt or excess amounts of proteinase inhibitors. Furthermore, this effect was not affected by either cytochalasin B or DNase I. These results therefore suggest that G-actin interacts with the enzyme molecule at the binding site(s) different from that involved in actin polymerization, and that it causes the activation of the soluble enzyme as a result of an allosteric alteration in the enzyme structure, thus giving rise to the possibility that cytoskeletal elements play an important role in the regulation of catecholamine synthesis as a factor modulating the activity of cytoplasmic tyrosine hydroxylase.

Long-term culture of avian embryonic cells in vitro.

The pH of the embryonic blood, one of the most important environmental factors for embryonic cells, was found to range from 8.1 to 8.5 in chick embryos until 108 h after incubation. Based on these results, the culture medium adjusted to pH 8.0 was used to culture embryonic chick and quail cells. They were easily subcultured for a long period of time at pH 8.0. This pH culture condition may have wide application for manipulating embryonic cells or tissues and establishing cell lines from avian embryos.

Chlorophyll treatment combined with photostimulation increases glycolysis and decreases oxidative stress in the liver of type 1 diabetic rats

Photodynamic therapy (PDT) promotes cell death, and it has been successfully employed as a treatment resource for neuropathic complications of diabetes mellitus (T1DM) and hepatocellular carcinoma. The liver is the major organ involved in the regulation of energy homeostasis, and in pathological conditions such as T1DM, changes in liver metabolic pathways result in hyperglycemia, which is associated with multiple organic dysfunctions. In this context, it has been suggested that chlorophyll-a and its derivatives have anti-diabetic actions, such as reducing hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, but these effects have not yet been proven. Thus, the biological action of PDT with chlorophyll-a on hepatic parameters related to energy metabolism and oxidative stress in T1DM Wistar rats was investigated. Evaluation of the acute effects of this pigment was performed by incubation of isolated hepatocytes with chlorophyll-a and the chronic effects were evaluated by oral treatment with chlorophyll-based extract, with post-analysis of the intact liver by in situ perfusion. In both experimental protocols, chlorophyll-a decreased hepatic glucose release and glycogenolysis rate and stimulated the glycolytic pathway in DM/PDT. In addition, there was a reduction in hepatic oxidative stress, noticeable by decreased lipoperoxidation, reactive oxygen species, and carbonylated proteins in livers of chlorophyll-treated T1DM rats. These are indicators of the potential capacity of chlorophyll-a in improving the status of the diabetic liver.

The effect of a single dose of osteoprotegerin in postmenopausal women.

Osteoprotegerin (OPG), a tumor necrosis factor (TNF) receptor family member, is a critical regulator of bone resorption. It is an important inhibitor of the terminal differentiation and activation of osteoclasts. This randomized, double-blind, placebo-controlled, sequential dose escalation study was conducted in postmenopausal women to determine the effect of a single subcutaneous (s.c.) dose of OPG on bone resorption as indicated by the biochemical markers, urinary N-telopeptide (NTX) and deoxypyridinoline (DPD), which are stable collagen degradation products. NTX levels decreased within 12 h after OPG administration. At the highest dose administered (3.0 mg/kg), a mean percent decrease in NTX of approximately 80% was observed 4 days after dosing. Six weeks after dosing a mean decrease of 14% in NTX was observed. The levels of bone-specific alkaline phosphatase (BSAP), a marker of bone formation, did not change for approximately 3 weeks after dosing. Thereafter, a modest decrease, reaching approximately 30% at 6 weeks, was observed in the 3.0-mg/kg dose group. The rapid decrease from baseline in NTX and delayed decrease in BSAP indicated that OPG acted primarily on osteoclasts to decrease bone resorption. OPG injections are well tolerated. This study, for the first time, indicates that a single s.c. injection of OPG is effective in rapidly and profoundly reducing bone turnover for a sustained period and that OPG therefore may be effective in treatment of bone diseases characterized by increased bone resorption such as osteoporosis.

Phorbol ester stimulates catecholamine synthesis in isolated bovine adrenal medullary cells.

In isolated bovine adrenal medullary cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, stimulated [14C]catecholamine synthesis from [14C]tyrosine, but not from [14C]DOPA. This stimulatory effect of TPA on [14C]catecholamine synthesis was not dependent upon extracellular Ca2+, and TPA did not affect the uptake of 45Ca2+ or the release of catecholamine by the cells. TPA also did not affect the intracellular cyclic AMP (cAMP) level. 4 alpha-Phorbol 12, 13-didecanoate, which is not an activator of protein kinase C, did not stimulate the synthesis of [14C]catecholamine from [14C]tyrosine. The stimulatory effect of TPA on [14C]catecholamine synthesis was additive with that of carbamylcholine, but not with that of dibutyryl cAMP (DB-cAMP). From these results, it was suggested that protein kinase C is involved in the regulation of tyrosine hydroxylase activity and that this regulatory mechanism might be similar to that involving cAMP.

Electron spin resonance analysis of heme-nitrosyl and reduced iron-sulfur centered complexes in allogeneic, heterotopic cardiac transplants: effects of treatment with pyrrolidine dithiocarbamate.

Inhibition of inducible nitric oxide synthase (iNOS) prolongs allograft survival suggesting a role for nitric oxide (.NO) in allograft rejection. Induction of iNOS is regulated by the oxidant-sensitive, nuclear factor kappa B (NF-kappaB) in many cell types. In the present study using electron spin resonance (ESR) spectroscopy, we evaluated whether pyrrolidine dithiocarbamate (PDTC), a metal chelator and antioxidant, might limit .NO production during the development of rejection in cardiac allografts. We performed either isogeneic (Lewis to Lewis) or allogeneic (Wistar-Furth to Lewis) heterotopic abdominal cardiac transplantation. Allograft recipients received daily injections of PDTC or aminoguanidine (a known inhibitor of iNOS). At postoperative days 4 or 6, grafted and native hearts of transplant recipients were flushed with cardioplegic solution to remove blood contamination. ESR data of allografts revealed a triplet nitrogen signal (aN=17.5 G) and centered at g=2.012 and an additional broad signal at g=2.08. This signal was not seen in either isografts or native hearts of either isograft or allograft recipients. Based upon these parameters, these signals are attributed to nitrosomyoglobin. This signal was inhibited by treatment with aminoguanidine or PDTC. Under these conditions, PDTC also prolonged graft survival from 6.6+/-0.2 to 11.7+/-0.3 days. Thus, it is conceivable that nitrosylmyoglobin formation precedes rejection in cardiac allografts and inhibition of nitrosomyoglobin with agents such as PDTC contribute to improved graft survival.

Myocardial nuclear factor-kappaB activity and nitric oxide production in rejecting cardiac allografts.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a rapid response transcription factor for genes whose products are critical for inflammation and immunity. In a rat model of heterotopic cardiac transplantation, we studied NF-kappaB DNA binding activity and nitric oxide (.NO) production in untreated allografts and whether inhibition of NF-kappaB suppresses .NO production and prolongs graft survival. METHODS: In allograft recipients and isograft controls, NF-kappaB was assayed by electrophoretic mobility shift assay, daily from transplant until rejection. Myocardial .NO was directly detected in explanted allografts by electron spin resonance spectroscopy on day 6 after transplant. The potent inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC; 250 mg/kg s.c.) was administered daily from transplant until day of rejection. The extent of graft lymphocytic infiltrate was assessed by routine hematoxylin and eosin staining. Immunohistochemical staining of NF-kappaB was per formed to identify the cell type responsible for NF-kappaB activity. RESULTS: A time-dependent increase in myocardial NF-kappaB activity was seen in untreated allografts as compared with isografts as determined by PhosphorImage analysis. Peak NF-kappaB activity occurred in allografts on day 4 with a ninefold increase as compared with isografts (24.0+/-3.7% vs. 2.7+/-0.5; P<0.05). On posttransplant day 6, electron spin resonance spectroscopy analysis of allografts demonstrated .NO identified by a triplet nitrogen signal centered at g=2.012 with hyperfine splitting of 17.5 Gauss, which is consistent with nitrosoheme formation and low-field signals at g=2.08 and g=2.03 consistent with nitrosomyoglobin. These signals were not seen in native hearts of allograft recipients. With PDTC administration, a threefold decrease in NF-kappaB activity within the transplanted heart was observed on posttransplant day 5 as compared with untreated allografts (9.7+/-1.6% vs. 23.5+/-2.5%; P<0.01). PDTC prolonged graft survival as compared with untreated allografts (11.7+/-0.3 vs. 6.6+/-0.2 days; P<0.05) and reduced the intensity of the nitrosoheme and nitrosomyoglobin signals. Allograft mononuclear cell infiltrate correlated with peak NF-kappaB activity with peak infiltrate on posttransplant day 4. PDTC treatment had no effect on the extent of infiltrate. Immunohistochemical staining localized NF-kappaB to the infiltrating mononuclear cells on posttransplant day 5. CONCLUSION: These data support a role for NF-kappaB in allograft rejection.

Stimulation by vasoactive intestinal polypeptide of catecholamine synthesis in isolated bovine adrenal chromaffin cells. Possible involvement of protein kinase C.

In isolated bovine adrenal medullary cells, vasoactive intestinal polypeptide (VIP) stimulated 14C-catecholamine synthesis from 14C-tyrosine, but not from 14C-DOPA. This stimulatory effect of VIP on 14C-catecholamine synthesis was not dependent upon extracellular Ca2+. VIP did not affect the intracellular cyclic AMP (cAMP) level. The stimulatory effect of VIP on 14C-catecholamine synthesis was additive with that of carbamylcholine, which was dependent upon extracellular Ca2+, but not with that of phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C. Moreover, 1-(isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, inhibited not only TPA-stimulated, but also VIP-stimulated 14C-catecholamine synthesis from 14C-tyrosine. These results suggested that VIP stimulated catecholamine synthesis by activation of tyrosine hydroxylase and that protein kinase C was involved in this stimulatory mechanism.

Myosin light-chain kinase inhibitor, 1-(5-chlornaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibits catecholamine secretion from adrenal chromaffin cells by inhibiting Ca2+ uptake into the cells.

For determination of whether myosin light-chain kinase (MLCK) is involved in the secretory mechanism of adrenal chromaffin cells, the effect of a preferential inhibitor of the enzyme, 1-(5-chlornaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), on catecholamine secretion from cultured bovine adrenal chromaffin cells was studied. ML-9 did not affect basal catecholamine secretion, but inhibited catecholamine secretion stimulated by acetylcholine, high K+, veratridine or palytoxin. At similar concentrations to those inhibiting the secretion of catecholamine, ML-9 also inhibited increased [45Ca]2+ uptake by the cells induced by these stimulants. However, it did not inhibit catecholamine secretion induced by the Ca2+ ionophore A23187. Moreover, it did not affect catecholamine secretion from digitonin-permeabilized cells induced by a micromolar Ca2+ concentration in the presence of Mg ATP. These results indicate that ML-9 inhibits catecholamine secretion from adrenal chromaffin cells by inhibiting the transmembrane Ca2+ uptake mechanism, but not by inhibiting the intracellular Ca2+-dependent mechanism. The possible role of MLCK in stimulus-secretion coupling in adrenal chromaffin cells is discussed.

Characterization of palytoxin-induced catecholamine secretion from cultured bovine adrenal chromaffin cells. Effects of Na(+)- and Ca(2+)-channel blockers.

The effect of palytoxin (PTX), a potent marine toxin, on catecholamine (CA) secretion from cultured bovine adrenal chromaffin cells was examined. PTX at concentrations of over 10(-10) M induced CA secretion concentration-dependently. About 40-50% of the total cellular CA was secreted during 20-min incubation with 3 x 10(-8) M PTX. PTX-induced CA secretion was dependent on both extracellular Na+ and Ca2+. PTX caused increases in [22Na](+)- and [45Ca](2+)-influxes into the cells. Increase in [22Na](+)-influx was observed at concentrations of over 10(-11) M PTX and was maximal at 10(-10) M PTX and then gradually decreased at higher concentrations that induced [45Ca](2+)-influx and CA secretion. On the other hand, increase in [45Ca](2+)-influx was observed at concentrations of over 10(-10) M PTX and increased with increase in concentration of PTX. This concentration-response curve for PTX-induced [45Ca](2+)-influx was similar to that for PTX-induced CA secretion. The CA secretion and [22Na](+)- and [45Ca](2+)-influxes induced by PTX were not affected by tetrodotoxin (TTX), but were significantly inhibited by quinidine and aprindine(mexiletine), antiarrythmic drugs known to block Na(+)-channels. Ca(2+)-channel blockers such as nifedipine, verapamil, Co2+, Cd2+, inhibited both CA secretion and [45Ca](2+)-influx induced by PTX. These results indicate that PTX-induced CA secretion is mediated by activation of Na(+)-dependent, TTX-insensitive voltage-dependent Ca(2+)-channels, and is inhibited by quinidine and aprindine through their inhibitory effects on the Na(+)- and Ca(2+)-influxes into the cells induced by PTX.