Anticholinesterase effect of the organophosphorus and pyrethroid insecticides to some phytophages species,their predators and parasites

It has been determined catalytic activity of cholinesterases for several insect species (Apanteles glomeratus L., Coccinella septempunctata L, Rhopalosiphumpadi L. and Pieris brassicae L.) that varies in norm from 57 to 199 mmol/hour per 1 gr. It has been calculated the constants of bimolecular interaction (K_ii) for insecticides Aztek, Mavric and Bi‐58 new with cholinesterase of the insects. It occurred to be the most sensitivity to Bi‐58 new is peculiar to this ferment in Coccinella 7‐punctata L. K_ii (4.54 ± 0.23) 10⁴; whereas cholinesterase of Rhopalosiphum padi L. is the least sensitive to Aztek ‐ K_ii (2.9 ± 0.14) 10⁵. Determination of anticholinesterase action coefficient has revealed the value of this indice 118.9 for the preparation Aztek in the system "Rhopalosiphum padi ‐Coccinella 7‐punctata”;. Supposedly, anticholinesterase activity of Aztek is the base of its mechanism action on above‐mentioned species of insects.     

Anticholinesterase activity of a new carbamate, heptylphysostigmine, in view of its use in patients with Alzheimer-type dementia

The anticholinesterase activity of a new carbamate, heptylphysostigmine, was studied in vitro. This compound is a competitive inhibitor of acetylcholinesterase (or true cholinesterase) having Ki = (1 +/- 0.5) X 10(-7) M. The inhibition was instantaneous at the onset and did not diminish with prolonged incubation of the drug and enzyme.     

Behavior of triatomines (Hemiptera: Reduviidae) vectors of Chagas' disease. II. Influence of feeding, lighting and time of day on the number of matings, mating speed and duration of copulation of Panstrongylus megistus (Burm, 1835) under laboratory conditions

To determine the influence of feeding, lighting and time of day on the copulating behavior of Panstrongylus megistus, 480 insect pairs were divided into four groups of 120 each and tested in the following respective situations: without food deprivation (F.D.), with five days of F.D., with ten days of F.D., and with 20 days of F.D. The tests were performed between 9:00 a.m. to 12:00 a.m. and 7:00 p.m. to 10:00 p.m., with light (700-1400 lux) and in the dark (1.4-2.8 lux) and behavior was recorded by the time sampling technique. Mating speed (MS) and duration of copulation (DC) were also calculated for each situation. The maximum frequency of copulation was observed after five days of F.D., at night, in the dark (n = 16), and the minimum was observed for recently-fed pairs, at night, with light (n = 4). Males approached females more often than females approached males. MS was lowest in pairs with twenty days of F.D., at night, with light (mean = 23.0 +/- 16.0 minutes), and highest in recently-fed pairs, during the day, with light (mean = 2.9 +/- 2.5 minutes). DC was shortest in recently-fed insects, during the day, in the dark (mean = 23.5 +/- 6.7 minutes), and longest in recently-fed animals, at night, in the dark (mean = 38.3 +/- 6.9 minutes).     

Determination of trace quantities of anticholinesterase pesticides

A procedure has been described which is applicable to the screening of large numbers of samples for anticholinesterase activity. The method is sufficiently sensitive to detect cholinesterase depression due to as little as 30 ppt paraoxon; 3 ppb malaoxon or 2 ppm of carbaryl in either ethylene glycol or water. While the method does not allow the absolute differentiation between compounds which might be present, some classification is possible, i.e., for compounds which must be activated before they possess anticholinesterase activity.     

The activation of diazinon by ganglia of the cockroach Periplaneta americana L. and its action on nerve conduction and cholinesterase activity

When sixth abdominal ganglia of the cockroach Periplaneta americana were irrigated continuously with diazinon solution in situ, its effects on nerve conduction and cholinesterase activity closely resembled those of diazoxon; spontaneous activity and after-discharge increased until conduction was blocked, which happened while some cholinesterase was still uninhibited. The symptoms were only slightly relieved by irrigating ganglia with saline. Though the LD50's of diazinon and diazoxon applied topically to adult male P. americana were similar (2.5 ± 0.33 and 4.5 ± 0.38 μig. per insect), diazoxon was about 300 times more active than diazinon against nerve function and cholinesterase activity in the sixth abdominal ganglion. This is probably because in the nerve preparations contact between the insecticide and the tissues surrounding the nerve cord, which in whole insects convert diazinon, a thionophosphate, into its phosphate analogue diazoxon, a more active anticholinesterase, was minimized. Indeed, taking into account the evidence of workers who previously compared in vitro the anticholinesterase activities of several thionophosphates with those of their phosphate analogues and found the phosphates much more active, the effect of diazinon on cholinesterase activity and nerve function in our experiments was unexpectedly great. By applying diazinon to nerve cords with SKF 525-A, a compound likely to prevent oxidation of diazinon to diazoxon, an attempt was therefore made to decide whether diazinon directly affected nerve conduction or whether the effect resulted either from its conversion to diazoxon within the nerve tissue or from impurities in the diazinon used. Results were inconclusive, for SKF 525-A (p-diethylaminoethyl diphenylpropylacetate hydrochloride) not only failed to prevent the inhibition of cholinesterase, but interfered with the action of both diazinon and diazoxon on nerve conduction, and itself affected nerve conduction when applied alone. The possibility that diazinon is itself a mild anticholinesterase was not excluded. SKF 525-A applied to sixth abdominal ganglia at 2 × 10^-4M blocked conduction from cereal nerves to giant fibres in 50–97 min. and at 4 × 10^-5M decreased the post-synaptic response; applied to giant fibres at 2 × 10^-4M it blocked conduction in 90–208 min. The effects of the larger concentration were not completely reversible. Although SKF 525-A has been widely used to study the metabolism of drugs, its direct effects on conduction in nerve axons seem not to have been noted previously.     

Haloalkane dehalogenases: steady-state kinetics and halide inhibition

The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (XaDHL) and Rhodococcus rhodochrous (RrDHL) have been compared using a pH-indicator dye assay. In contrast to XaDHL, RrDHL is efficient toward secondary alkyl halides. Using steady-state kinetics, we have shown that halides are uncompetitive inhibitors of XaDHL with 1, 2-dichloroethane as the varied substrate at pH 8.2 (Cl-, Kii = 19 +/- 0.91; Br-, Kii = 2.5 +/- 0.19 mM; I-, Kii = 4.1 +/- 0.43 mM). Because they are uncompetitive with the substrate, halide ions do not bind to the free form of the enzyme; therefore, halide ions cannot be the last product released from the enzyme. The Kii for chloride was pH dependent and decreased more than 20-fold from 61 mM at pH 8.9 to 2.9 mM at pH 6.5. The pH dependence of 1/Kii showed simple titration behavior that fit to a pKa of approximately 7.5. The kcat was maximal at pH 8.2 and decreased at lower pH. A titration of kcat versus pH also fits to a pKa of approximately 7.5. Taken together, these data suggest that chloride binding and kcat are affected by the same ionizable group, likely the imidazole of a histidyl residue. In contrast, halides do not inhibit RrDHL. The Rhodococcus enzyme does not contain a tryptophan corresponding to W175 of XaDHL, which has been implicated in halide ion binding. The site-directed mutants W175F and W175Y of XaDHL were prepared and tested for halide ion inhibition. Halides do not inhibit either W175F or W175Y XaDHL.     

Effect of calcium on rat intestinal alkaline phosphatase activity and molecular aggregation

Two fractions of rat intestinal alkaline phosphatase (IAP) were detected by Western blot: 168 +/- 6 and 475 +/- 45 kDa. The low molecular weight fraction constitutes 43% of the isolated proteins exhibiting 82% of the enzymatic activity, and a heavier fraction constitutes 57% of the isolated proteins and has 18% of the enzymatic activity. Calcium produced an increase of the 475-kDa form to the detriment of the 168-kDa form. This work also describes the kinetic and structural changes of IAP as a function of calcium concentration. With [Ca2+] < 10 mmole/L, the Ca(2+)-IAP interaction fitted a binding model with 7.8 +/- 4.4 moles of Ca2+ /mole of protein, affinity constant = 19.1 +/- 8.4 L/mmole, and enzymatic activity increased as a linear function of [Ca2+] (r = 0.946 p < 0.01). On the other hand, with [Ca2+] > 10 mmole/L the data did not fit this model and, the enzymatic activity decreased as a function of [Ca2+] (r = - 0.703 p < 0.05).     

Reversibility of the effects on blood cells,lipids, liver function and hormones in former anabolic-androgenic steroid abusers

BACKGROUND: In contrast to the acute effects of anabolic-androgenic steroid (AAS) abuse, the long-term risk profile of former long-term abusers (ExA) is less clear. METHODS: Blood parameters of 32 male bodybuilders and powerlifters were studied. Fifteen ExA had not been abusing AAS for at least 12-43 months on average (mean dosage 700 mg for 26 weeks per year over 9 years), 17 athletes (A) were still abusing AAS (750 mg for 33 weeks per 8 years). FINDINGS: Hemoglobin (+5%), leucocytes (+33%) and platelets (+38%) were significantly higher in A. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were higher, cholinesterase activity (CHE) lower in A (65+/-55, 38+/-27 and 3719+/-1528U/l) compared to ExA (24+/-10, 18+/-11 and 6345+/-975U/l; each P<0.001) with normal values for gamma-glutamyl transpeptidase (gamma-GT) and bilirubin. ALT, AST and CHE correlated significantly with the extent (duration and weekly dosage, expressed as a point score) of AAS abuse in A (r=0.68, 0.57 and -0.62; each P<0.01). Total and LDL-cholesterol were similar, HDL-cholesterol was distinctly lower in A than in ExA (17+/-11 and 43+/-11 mg/dl; P<0.001) and correlated negatively with the extent of AAS abuse (r=-0.50; P<0.05). Testosterone and estradiol were significantly higher, while LH, FSH and the sexual-hormone-binding (SHB) protein were lower in A than in ExA (each P<0.001). Two ExA had testosterone levels below the normal range. INTERPRETATION: The alterations in cell counts, HDL-cholesterol, liver function and most hormones of the pituitary-testicular axis induced by a long-term abuse of AAS were reversible after stopping the medication for over 1 year. In some ExA, an increased ALT activity and a depressed testosterone synthesis were found.     

Tissue distribution and characterization of cholinesterase activity in six earthworm species

To validate cholinesterase activity as a biomarker of pesticide exposure, we characterized the tissue distribution (whole body, nervous tissue and crop/gizzard), activity at two seasons of cholinesterase in six different species of earthworms collected in an unpolluted field: Lumbricus terrestris, Lumbricus castaneus, Aporrectodea nocturna, Aporrectodea caliginosa, Allolobophora chlorotica and Aporrectodea rosea. The major part of total cholinesterase activity was found in the nervous tissue while activity in crop/gizzard was weak. The level of the total cholinesterase activity was stable for each species considered throughout the year (spring and autumn). Lumbricus species exhibited three-fold higher specific activity than the others (0.086+/-0.015 U mg(-1) and 0.235+/-0.036 U mg(-1) for Allolobophora or Aporrectodea, and Lumbricus species respectively). This stability of the base level makes cholinesterase activity a useful biomarker for monitoring effects of pesticide under natural conditions. Cholinesterase activity was characterized using different substrates and inhibitors. It seems likely that the cholinesterases are acetylcholinesterases in most species investigated as they preferentially hydrolyzed acetylthiocholine and were inhibited by eserine, but not by tetraisopropyl pyrophoramide (iso-OMPA). Characterization of cholinesterase from Allolobophora chlorotica is uncertain and it cannot be classified as a true AChE.     

Are the dynamic response characteristics of brachial artery flow-mediated dilation sensitive to the magnitude of increase in shear stimulus?

The purpose of this study was to determine the dynamic characteristics of brachial artery dilation in response to step increases in shear stress [flow-mediated dilation (FMD)]. Brachial artery diameter (BAD) and mean blood velocity (MBV) (Doppler ultrasound) were obtained in 15 healthy subjects. Step increases in MBV at two shear stimulus magnitudes were investigated: large (L; maximal MBV attainable), and small (S; MBV at 50% of the large step). Increase in shear rate (estimate of shear stress: MBV/BAD) was 76.8 +/- 15.6 s(-1) for L and 41.4 +/- 8.7 s(-1) for S. The peak %FMD was 14.5 +/- 3.8% for L and 5.7 +/- 2.1% for S (P < 0.001). Both the L (all subjects) and the S step trials (12 of 15 subjects) elicited a biphasic diameter response with a fast initial phase (phase I) followed by a slower final phase. Relative contribution of phase I to total FMD when two phases occurred was not sensitive to shear rate magnitude (r(2) = 0.003, slope P = 0.775). Parameters quantifying the dynamics of the FMD response [time delay (TD), time constant (tau)] were also not sensitive to shear rate magnitude for both phases (phase I: TD r(2) = 0.03, slope P = 0.376, tau r(2) = 0.04, slope P = 0.261; final phase: TD r(2) = 0.07, slope P = 0.169, tau r(2) = 0.07, slope P = 0.996). These data support the existence of two distinct mechanisms, or sets of mechanisms, in the human conduit artery FMD response that are proportionally sensitive to shear stimulus magnitude and whose dynamic response is not sensitive to shear stimulus magnitude.     

Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells

The counterregulatory action of catecholamines on insulin-stimulated glucose transport and its relation to glucose transporter phosphorylation were studied in isolated rat adipose cells. Plasma membranes exhibiting reduced glucose transport activity were prepared as described previously (Joost, H. G., Weber, T. M., Cushman, S. W., and Simpson, I. A. (1986) J. Biol. Chem. 261, 10033-10036) from cells treated with insulin, and subsequently with isoproterenol and adenosine deaminase. In these membranes, transporter affinity for cytochalasin B binding was significantly reduced (KD = 133.5 +/- 14 versus 89.8 +/- 11 nM, means +/- S.E.) with no change in number of sites or immunoreactivity of the transporter on Western blots. Reconstituted plasma membrane transport was significantly lower with isoproterenol treatment (0.50 +/- 0.12 versus 0.97 +/- 0.27 nmol/mg protein/10 s). In contrast, transport activity reconstituted from corresponding intracellular transporters (from low density microsomes) was unchanged (5.4 +/- 2.2 versus 6.9 +/- 1.2 nmol/mg protein/10 s). Thus, the intrinsic activity change of the transporter produced by catecholamines appears to reflect a structural modification that is confined to the plasma membrane and not recycled into the intracellular compartment. In cells equilibrated with [32P]phosphate, neither insulin nor isoproterenol induced [32P]phosphate incorporation into the glucose transporter immunoprecipitated from plasma membranes. Conversely, phorbol 12-myristate 13-acetate stimulated significant incorporation of [32P]phosphate into the glucose transporter in insulin-stimulated cells without any change in plasma membrane transport activity or transporter concentration. Thus, the phosphorylation state of the glucose transporter does not seem to be involved in either signaling transporter translocation or triggering changes in transporter intrinsic activity.     

Combined effect of short-term dehydration and sublethal acute oral dicrotophos exposure confounds the diagnosis of anticholinesterase exposure in common quail (Coturnix coturnix) using plasma cholinesterase activity

Common Quail (Coturnix coturnix) were subjected to controlled and replicated experiments in the summer of 2008 to investigate the effects of short-term dehydration on cholinesterase activity in brain and plasma and the interaction between dehydration and exposure to the organophosphorus pesticide dicrotophos in these same tissues. Our objective was to determine if dehydration could confound the diagnosis of anticholinesterase exposure using inhibition of cholinesterase activity in quail tissues. The effect of dehydration was quantified using measures of plasma osmolality and hematocrit. Dicrotophos exposure caused significant inhibition of cholinesterase activity in brain, while the effects of dehydration and interaction were not significant. Dehydration caused significant duration-dependent increases in plasma osmolality and hematocrit. Dehydration also caused a significant increase in plasma cholinesterase activity. Variation in the change in plasma cholinesterase activity in response to dehydration was significantly and positively correlated with dehydration-induced variation in both the change in plasma osmolality and the change in hematocrit. These correlations suggest that plasma cholinesterase activity in quail is not limited to plasma but occupies some larger pool of the extracellular fluid volume, and we suggest lymph is part of that pool. The effects of dehydration on plasma cholinesterase activity masked the inhibitory effects of dicrotophos. Here, the combination of dehydration and dicrotophos exposure produced plasma cholinesterase activity that was not significantly different from reference and pre-exposure values, confounding the diagnosis of anticholinesterase exposure in dehydrated, dicrotophos-exposed quail. A method to adjust plasma cholinesterase activities for the confounding effects of dehydration and enable the diagnosis of anticholinesterase exposure in dehydrated, dicrotophos-exposed quail was developed. Clinicians and practitioners responsible for the diagnosis of anticholinesterase exposure in birds are cautioned that dehydration, commonly observed in sick wildlife, may mask the effect of anticholinesterases on plasma cholinesterase activity.     

A comparative study on effects of normal versus elevated temperatures during preimaginal and young adult period on body weight and fat body content of mature Coccinella septempunctata and Harmonia axyridis (Coleoptera: Coccinellidae)

Two climate chamber experiments were performed to simulate the effects of global warming on life table parameters of coccinellids. We investigated the effects of two daily temperature profiles during preimaginal development (larval and pupal) and the young adult period (first 10 d) on body weight and fat body content of adult Coccinella septempunctata L. and Harmonia axyridis (Pallas) fed English grain aphids [Sitobion avenae (F.)] ad libitum: 1) normal, i.e., current daily temperatures in central Europe (T0: mean, 17.8°C; maximum, 21.8°C; minimum, 13.4°C) and 2) increased by 3K (T3: mean, 20.8°C; maximum, 25.5°C; minimum, 15.7°C). The first experiment was performed at the same temperatures (T0 or T3) during both periods to establish the responses of the two species to temperature. The second was conducted to identify the period (preimaginal or adult) in which the responses occurred and to confirm the results of the first experiment. Compared with normal temperatures (T0), elevated temperatures (T3) resulted in significant decreases in preimaginal development time and increases in aphid consumption rates in both species. C. septempunctata (10-d-old adults) had the highest weights when reared at T3, H. axyridis at T0. C. septempunctata was significantly heavier than H. axyridis in most cases, particularly in females. The body fat content of C. septempunctata was higher than that of H. axyridis at T0 and T3 temperatures. At T3 temperatures, fat accumulation in C. septempunctata increased, whereas that in H. axyridis remained relatively low. Body weight and fat body content of 10-d-old adults of both species seemed to be determined by temperature conditions during preimaginal development.     

O,O-dialkyl-S-bromomethylthiophosphates--inhibitors of mammalian choline- and carboxyl esterases: structure-activity relationship

The interaction kinetics of potential pesticides, O,O-dialkyl S-bromomethylthiophosphates (RO)2P(O) SCH2Br (R = Et, i-Pr, n-Pr, n-Bu, or n-Am) with acetylcholinesterase, butyryl cholinesterase, and carboxyl esterase from warm-blooded animals was studied. All the compounds irreversibly inhibit these esterases, with k1 (M-1 min-1) being 1.8 x 10(4) - 1.9 x 10(6) for acetylcholinesterase, 2.0 x 10(6) - 4.1 x 10(7) for the more sensitive butyryl cholinesterase, and 2.3 x 10(7) - 2.3 x 10(8) and higher for the most sensitive carboxyl esterase. By using the Hansch and Kubinyi technique of multiple regression analysis, we quantitatively analyzed the relationship between the structure and inhibiting activity of these substances toward acetylcholinesterase and butyryl cholinesterase. Hydrophobic interactions were found to be important for the inhibition of both enzymes but are more pronounced in the case of butyryl cholinesterase. On the other hand, steric factors were much more significant in the inhibition of acetylcholinesterase. For both enzymes, the steric hindrances affect the phosphorylation stage of the enzyme.     

A novel butyrylcholinesterase from serum of Leporinus macrocephalus, a Neotropical fish

We show here that serum of piaussu, a Neotropical characin fish, has the highest butyrylcholinesterase activity so far described for humans and fish. To clarify whether this cholinesterase could protect piaussu against anticholinesterase pesticides by scavenging organophosphates, we purified it 1700-fold, with a yield of 80%. Augmenting concentrations (from 0.01 to 20 mM) of butyrylthiocholine activated it. The pure enzyme was highly inhibited by chlorpyriphos-oxon (ki=10,434x10(6) M-1 min-1) and by the specific butyrylcholinesterase inhibitor, isoOMPA (ki=45.7x10(6) M-1 min-1). Electrophoresis of total serum and 2-D electrophoresis of the purified cholinesterase showed that some enzyme molecules could circulate in piaussu serum as heterogeneously glycosylated dimers. The enzyme's N-terminal sequence was similar to sequences found for butyrylcholinesterase from sera of other vertebrates. Altogether, our data present a novel butyrylcholinesterase with the potential of protecting a fish from poisoning by organophosphates.     

Hematological, protein electrophoresis and cholinesterase values of free-living nestling peregrine falcons in Spain

Protein electrophoresis, hematological and cholinesterase values were determined in 32 nestling free-living peregrine falcons (Falco peregrinus) (15- to 27-days-old) in order to establish normal reference values for this population. The following values (mean +/- SD) were observed: prealbumin 0.31 +/- 0.04 g/dl, albumin 1.25 +/- 0.06 g/dl, alpha1 and alpha2-globulin 0.23 +/- 0.02 and 0.16 +/- 0.02 g/dl respectively, beta-globulin 1.02 +/- 0.05 g/dl, gamma-globulin 0.060 +/- 0.08 g/dl, total protein 3.79 +/- 0.18 g/dl, 21.26 +/- 1.30 white blood cells/microl (1 x 10(3)), 2.17 +/- 0.07 red blood cells/microl (1 x 10(6)), packed cell volume 37.58 +/- 0.82%, hemoglobin 20.96 +/- 0.29 g/dl, heterophils 61.14 +/- 2.50% and cholinesterase 1,184 +/- 75 IU/L. There were no difference in any of these parameters among males and females. The hematological values obtained could be considered as representative values in free-living nestling peregrine falcons.     

Role of enhanced detoxication in a deltamethrin-resistant population of Triatoma infestans (Hemiptera, Reduviidae) from Argentina

Deltamethrin and other pyrethroids have been extensively used in Argentina since 1980, for the chemical control of Triatoma infestans Klug (Hemiptera: Reduviidae). Recently, resistance to deltamethrin was detected in field populations by the survival of bugs exposed by topical application to the diagnostic dose estimated on the CIPEIN susceptible strain. Results of the current study showed low resistant ratios (RRs) to deltamethrin for the resistant populations (RR ranged from 2.0 for San Luis colony to 7.9 for Salta colony). Biochemical studies were made on the most resistant colony (Salta) and the susceptible strain (CIPEIN), in order to establish the importance of degradative mechanisms as a cause of the detected resistance. Esterase activity was measured on 3 days old first instars through phenylthioacetate and a-naphtyl acetate activities. The results showed a significant difference in no cholinesterase esterase activity from susceptible (7.6 +/- 0,7 micro M S./i.min.) and Salta resistant colony (9.5 +/- 0.8 microM S./i.min.). Cytochrome p450 mono-oxygenase (p450) activity was measured on individual insects through ethoxycoumarine deethylase (ECOD) activity using a fluorescence microplate reader. The dependence of ECOD activity on age and body region of the nymphs, and pH and time of incubation were studied in order to optimize the measurement. As a result, comparative studies were performed on abdomens of 2 days old first instars at pH 7.2 and 4 h incubation time. ECOD activity of first nymphs was significantly lower in the susceptible colony (61.3 +/- 9.08 pg ECOD/ insect) than in the resistant one (108.1+/- 5.7 pg ECOD/ insect). These results suggest that degradative esterases (no-cholinesterase) and mono-oxygenases cytochrome p450, play an important role in the resistance to deltamethrin in Salta colony from Argentina.     

NMR measurement of cytosolic free calcium, free magnesium, and intracellular sodium in the aorta of the normal and spontaneously hypertensive rat

We have utilized multinuclear NMR spectroscopy to examine the relationship between cytosolic free Ca2+ ([Ca2+]in), free Mg2+ ([Mg2+]in) and intracellular Na+ ([Na+]in) levels of the intact thoracic aorta and primary hypertension using the Wistar-Kyoto and Sprague-Dawley rats as controls and the spontaneously hypertensive rat as a model for genetic hypertension. Cytosolic free [Ca2+] was measured using 19F NMR of the intracellular Ca2+ indicator 5,5'-difluoro-1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, free [Mg2+] using the 31P resonances of intracellular ATP, and intracellular [Na+] by 23Na NMR in combination with the extracellular shift reagent dysprosium tripolyphosphate. We have found that both the [Na+]in and [Ca2+]in levels were significantly increased in the hypertensive animals relative to normotensive controls (p less than 0.01). Mean systolic blood pressures (using tail cuff method) of control and hypertensive rats were 123 +/- 8 mm Hg (mean +/- 2 S.E., n = 7) and 159 +/- 6 mm Hg (mean +/- 2 S.E., n = 7), respectively. [Na+]in and [Ca2+]in were 21.9 +/- 6.4 mM (mean +/- 2 S.E., n = 7) and 277 +/- 28 nM (mean +/- 2 S.E., n = 5) for the spontaneously hypertensive rats versus 10.1 +/- 1.8 mM (mean +/- 2 S.E., n = 7) and 151 +/- 26 nM (mean +/- 2 S.E., n = 5) for control rats, respectively. A slight difference observed between intracellular free Mg2+ levels in hypertensives (180 +/- 38 microM, mean +/- 2 S.E., n = 4) and controls (246 +/- 76 microM, mean +/- 2 S.E., n = 4) was not statistically significant (p greater than 0.1). These data indicate alterations in the cell membrane ion transport function of the aortic smooth muscle in primary hypertension.     

Expression of airway contractile properties and acetylcholinesterase activity in swine

We studied the effect of maturation on contractile properties of tracheal smooth muscle from seventeen 2-wk-old swine (2ws) and fifteen 10-wk-old swine (10ws) in situ and in vitro. The response to parasympathetic stimulation was studied in situ in isometrically fixed segments. Contraction was elicited at lower frequencies [half-maximal response to electrical stimulation (ES50) = 6.7 +/- 0.05 Hz] in 2ws than in 10ws (ES50 = 9.1 +/- 0.4 Hz; P less than 0.01). Despite substantial differences in morphometrically normalized cross-sectional area in 2ws (0.012 +/- 0.003 cm2) and 10ws (0.028 +/- 0.001 cm2; P less than 0.01), maximal active tension elicited by parasympathetic stimulation was similar (12.4 +/- 3.2 g/cm in 2ws vs. 13.3 +/- 2.3 g/cm in 10ws; P = NS). In separate in vitro studies in 25 tracheal smooth muscle strips from 10 swine, concentration-response curves generated with potassium-substituted Krebs solution (KCl) were similar in 2ws and 10ws. In 58 other strips (10 swine), maximal active force elicited with acetylcholine (ACh) in 2ws was significantly greater than for 10ws (P less than 0.001). Removal of the epithelium had no effect. However, cholinesterase inhibition with 10(-7) M physostigmine augmented the response to ACh in 10ws (P less than 0.02) but not 2ws. We demonstrate increased force generation and sensitivity to vagal stimulation in 2ws vs. 10ws, which corresponds to increased reactivity to ACh in vitro. The relative hyperresponsiveness in 2ws is specific for cholinergic response and is attenuated at least in part by maturation of the activity of acetylcholinesterase enzyme.     

Rhythmic expression, light entrainment and alpha-MSH modulation of rhodopsin mRNA in a teleost pigment cell line

To investigate whether teleost fish GEM-81 erythrophoroma cells were photosensitive, the cells were submitted to constant darkness (DD), 14 h of light and 10 h of darkness (14L:10D), and 10 h of light and 14 h of darkness (10L:14L). The doubling times (hours) were: DD 35.33+/-0.05; 14L:10D 67.85+/-0.04; and 10L:14D 49.60+/-0.08. In order to verify whether proliferation was dependent on light phase length, GEM-81 cells were submitted to 7L: 5D. The proliferation curves and doubling times were similar in 14L:10D and 7L:5D (respectively 69.44+/-0.03 and 67.85+/-0.04), suggesting that the cell cycle was regulated by the length of the light phase within 24 h, or by the light/dark ratio. We have also demonstrated the expression of Carassius retinal rhodopsin mRNA in GEM-81 cells, which cycles in a circadian rhythm, entrained by light. In addition, we showed that alpha-melanocyte stimulating hormone (alpha-MSH, 10(-10) to 10(-8) M), a conspicuous hormone that exerts mitogenic and melanogenic activity in most vertebrates, decreased rhodopsin mRNA in the first 3 days; after 4 days the inhibition was reversed, and after 5 days an increase in rhodopsin mRNA level was elicited. This is the first report of rhythmic expression of extra-ocular rhodopsin and its modulation by light and hormones.