Chronic treatment with D-chiro-inositol prevents autonomic and somatic neuropathy in STZ-induced diabetic mice.

AIM: D-chiro-inositol (DCI) has been shown to prevent and reverse endothelial dysfunction in diabetic rats and rabbits. The present study evaluates the preventive effect of DCI on experimental diabetic neuropathy (DN). METHODS: Streptozotocin-induced (STZ) diabetic mice were treated by oral gavage for 60 days with DCI (20 mg/kg/12 h) or saline (NaCl 0.9%; 0.1 ml/10 g/12 h; Diab) and compared with euglycaemic groups treated with saline (0.1 ml/10 g/12 h; Eugly). We compared the response of the isolated sciatic nerve, corpora cavernosa or vas deferens to electrical stimulation. RESULTS: The electrically evoked compound action potential of the sciatic nerve was greatly blunted by diabetes. The peak-to-peak amplitude (PPA) was decreased from 3.24 ± 0.7 to 0.9 ± 0.2 mV (p < 0.05), the conduction velocity (CV) of the first component was reduced from 46.78 ± 4.5 to 26.69 ± 3.8 ms (p < 0.05) and chronaxy was increased from 60.43 ± 1.9 to 69.67 ± 1.4 ms (p < 0.05). These parameters were improved in nerves from DCI-treated mice (p < 0.05). PPA in the DCI group was 5.79 ± 0.8 mV (vs. 0.9 ± 0.2 mV-Diab; p < 0.05) and CV was 45.91 ± 3.6 ms (vs. 26.69 ± 3.8 ms-Diab; p < 0.05). Maximal relaxation of the corpus cavernosum evoked by electrical stimulation (2-64 Hz) in the Diab group was 36.4 ± 3.8% compared to 65.4 ± 2.8% in Eugly and 59.3 ± 5.5% in the DCI group (p < 0.05). Maximal contraction obtained in the vas deferens was 38.0 ± 9.2% in Eugly and 11.5 ± 2.6% in Diab (decrease of 69.7%; p < 0.05), compared to 25.2 ± 2.3% in the DCI group (p < 0.05 vs. diabetic). Electron microscopy of the sciatic nerves showed prevention of neuronal damage. CONCLUSIONS: DCI has a neuroprotective action in both autonomic and somatic nerves in STZ-induced DN.

Lung injury caused by occupational exposure to particles from the industrial combustion of cashew nut shells: a mice model.

Cashew nut shells (CNS) is already used in the energy matrix of some industries. However, it is necessary to know the harmful health effects generated by exposure to pollutants of its combustion, especially in the workers exposed to industrial pollutants. In addition, it is known that the incidence of asthma grows among workers in industries, and due to its previously reported biological effects of anethole, these will also be objects of the present study. We used 64 Balb/C mice, randomly divided into eight groups. Groups were sensitized and challenged with saline or ovalbumin, then subjected to intranasal instillation of 30 µg PM4.0 (occupational exposure) from the combustion of CNS or saline, and then were subsequently treated with oral anethole 300 mg/kg or 0.1% Tween 80. Our results serve as a starting point for the development of public policies for the prevention of diseases in workers that are exposed to the pollutants coming from industries.

The essential oil of Hyptis crenata Pohl ex Benth. presents an antiedematogenic effect in mice.

Hyptis crenata, commonly known as "salva-do-Marajó", "hortelã-do-campo", and "hortelãzinha", is used in folk medicine in Northeast Brazil as tea or infusion to treat inflammatory diseases. Due to the pharmacological efficacy and the low toxicity of the essential oil of Hyptis crenata (EOHc), we decided to investigate the EOHc antiedematogenic effect in experimental models of inflammation. EOHc was administrated orally at doses of 10-300 mg/kg to male Swiss albino mice. Paw edema was induced by subcutaneous injection in the right hind paw of inflammatory stimuli (carrageenan, dextran, histamine, serotonin, and bradykinin) 60 min after administration of EOHc. EOHc significantly inhibited the induced edema. The inhibitory effect of EOHc on dextran-induced edema extended throughout the experimental time. For the 30, 100, and 300 mg/kg doses of EOHc, the inhibition was of 40.28±1.70, 51.18±2.69, and 59.24±2.13%, respectively. The EOHc inhibitory effect on carrageenan-induced edema started at 10 mg/kg at the second hour (h) and was maintained throughout the observation period. At 30, 100, and 300 mg/kg doses the inhibition started earlier, from 30 min. At the edema peak of 180 min, 56, 76, and 82% inhibition was observed for 30, 100, and 300 mg/kg doses, respectively. Additionally, the effect of EOHc on carrageenan-induced paw edema was influenced by the time of administration. The EOHc also inhibited myeloperoxidase activity. In conclusion, the EOHc showed a potent effect, both preventing and reversing the edema, consistent with its anti-inflammatory use in folk medicine.

Evaluation of Hypoglycemic Therapy Through Physical Exercise in n5STZ-Induced Diabetes Rats.

BACKGROUND: Diabetes mellitus is a syndrome with multiple etiologies involving insulin, in which there is a lack of production and/or loss of sensitivity to this hormone resulting in insulin resistance. Treatment and control of this disease requires changes in diet, use of medication, and lifestyle, such as physical activity. These modifications may compromise quality-of-life if there is no proper guidance for the treatment or alert to possible complications caused by the disease. METHODS: This study aimed to evaluate biochemical and hematological parameters, and to assess brain derived neurotrophic factor levels in diabetic Wistar rats submitted to chronic physical exercise. RESULTS: The results demonstrated an increase in plasma concentration of brain-derived neurotrophic factor (BDNF) in association with hyperglycemia reduction in diabetic animals. DISCUSSION: The results obtained suggest that there is a regulation of glucose homeostasis between peripheral tissues and the central nervous system. Exercise-induced BDNF also improved levels of glycemia, body weight, and dyslipidemia. In hematological evaluation, BDNF increase was positively correlated with an improvement in leukocyte parameters. Electrophoresis analyses demonstrated a reduction in levels of pro-inflammatory proteins, lipoprotein fractions, and albumin preservation in diabetic animals trained with elevated concentration of plasma BDNF. CONCLUSION: In conclusion, this study demonstrated that chronic exercise was able to elevate BDNF levels in plasma, which resulted directly in positive hypoglycemic activity in diabetic animals and a reduction of the metabolic syndrome associated with diabetes mellitus.

Expression of myo-inositol cotransporters in the sciatic nerve and dorsal root ganglia in experimental diabetes.

The transport of myo-inositol is the main mechanism for the maintenance of its high intracellular levels. We aimed to measure the mRNA and protein levels of myo-inositol cotransporters in the sciatic nerve (SN) and dorsal root ganglia (DRG) during experimental diabetes. Streptozotocin-induced (STZ; 4, 8, and 12 weeks; 65 mg/kg; ip) diabetic rats (DB) and age-matched euglycemic (E) rats were used for the analysis of mRNA and protein levels of sodium myo-inositol cotransporters 1, 2 (SMIT1, SMIT2) or H+/myo-inositol cotransporter (HMIT). There was a significant reduction in the mRNA levels for SMIT1 in the SN and DRG (by 36.9 and 31.0%) in the 4-week DB (DB4) group compared to the E group. SMIT2 was not expressed in SN. The mRNA level for SMIT2 was up-regulated only in the DRG in the DB4 group. On the other hand, the protein level of SMIT1 decreased by 42.5, 41.3, and 44.8% in the SN after 4, 8, and 12 weeks of diabetes, respectively. In addition, there was a decrease of 64.3 and 58.0% of HMIT in membrane and cytosolic fractions, respectively, in the SN of the DB4 group. In the DRG, there was an increase of 230 and 86.3% for SMIT1 and HMIT, respectively, in the DB12 group. The levels of the main inositol transporters, SMIT1 and HMIT, were greatly reduced in the SN but not in the DRG. SMIT-1 was selectively reduced in the sciatic nerve during experimental STZ-induced diabetes.

Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles.

Primordial follicles, the main source of oocytes in the ovary, are essential for the maintenance of fertility throughout the reproductive lifespan. To the best of our knowledge, there are no reports describing the effect of anethole on this important ovarian follicle population. The aim of the study was to investigate the effect of different anethole concentrations on the in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Randomized ovarian fragments were fixed immediately (non-cultured treatment) or distributed into five treatments: α-MEM+ (cultured control), α-MEM+ supplemented with ascorbic acid at 50 µg/mL (AA), and anethole at 30 (AN30), 300 (AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a significantly higher percentage of morphologically normal follicles was observed when anethole at 2000 µg/mL was used. For both culture times, a greater percentage of growing follicles was observed with the AN30 treatment compared to AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1 and 7 of culture resulted in significantly larger follicular diameter than in the cultured control treatment. Anethole at 30 µg/mL concentration at day 7 showed significantly greater oocyte diameter than the other treatments, except when compared to the AN2000 treatment. At day 7 of culture, levels of reactive oxygen species (ROS) were significantly lower in the AN30 treatment than the other treatments. In conclusion, supplementation of culture medium with anethole improves survival and early follicle development at different concentrations in the caprine species.

Vasorelaxant effect of the Lippia alba essential oil and its major constituent, citral, on the contractility of isolated rat aorta.

The Lippia alba (Mill.) N.E. Brown (Verbenaceae) species popularly known as lemon balm has sedative, analgesic and spasmolytic properties. This study aimed to evaluate the vasorelaxant effect of the L. alba essential oil (EOLa) and its major constituent, citral, rat on aorta. Isometric muscle contraction were induced by potassium (K 60 mM) or phenylephrine (PHE, 0.1 µM) in isolated aortic rings. EOLa and citral promoted a smooth muscle relaxant action, which was potentiated by the presence of the endothelium; PHE-induced contractions (0.1 µM) in aorta with endothelium, had EC50 values of 352.73 ± 19.39 µg/mL and 99.34 ± 7.2 µg/mL for EOLa and citral, respectively. In the presence of a nitric oxide synthase inhibitor, L-NAME, the EC50 values were 654.19 ± 10.46 µg/mL and 601.66 ± 10.922 µg/mL for EOLa and citral, respectively. EOLa and citral dose-dependently relaxed contractions induced by BAY-K 8644, a calcium channel agonist, and by Phorbol 12,13-dibutyrate an activator of protein kinase C. EOLa and citral produced a vasorelaxant effect in isolated aorta which was potentiated by the presence of endothelium. In summary, EOLa and citral, probably using several mechanisms of action, relaxed aortic smooth muscle with maximal pharmacologic efficacy.

Essential oil of Pterodon polygalaeflorus Benth attenuates nociception in mice.

Essential oils (EO) are volatile liquids responsible for the aroma of plants. Pterodon polygalaeflorus seeds have received widespread use in folk medicine for the treatment of inflammatory diseases. For this reason and because Pterodon polygalaeflorus seeds have great EO content, which is frequently pharmacologically active, the present study aimed to evaluate the antinociceptive effect of EO from Pterodon polygalaeflorus (EOPPgfl) and its acute toxic effects. The EEOPPgfl sample, which was extracted by steam distillation of the seeds, had a yield of 2.4% of the seeds weight and had, as major constituents, beta-elemene (48.19%), trans-caryophyllene (19.51%), and epi-bicyclosesquiphellandrene (12.24%). The EOPPgfl sample showed mild acute toxicity and its calculated median lethal dose (LD50) was 3.38 g/kg. EOPPgfl (20-60 mg/kg) showed antinociceptive activity as evidenced by several tests and inhibited writhing induced by acetic acid. The maximum effect was obtained with the 30 mg/kg dose and at 60 min after its administration. EOPPgfl also decreased formalin-induced nociception, as verified by the inhibition of the first and second phase of the formalin test. At 30 mg/kg, EOPPgfl also decreased thermally stimulated nociception. Nociception may be related to inflammatory and antiedematogenic activity and at doses ranging 10-100 mg/kg, EOPPgfl blocked dextran- and carrageenan-induced edema. The results demonstrated that EOPPgfl presented, at doses approximately 100 times smaller than LD50, an antinociceptive effect that probably was due to anti-inflammatory activities.

Effects of Lippia sidoides essential oil, thymol, p-cymene, myrcene and caryophyllene on rat sciatic nerve excitability.

Lippia sidoides Cham is a typical herb species of Northeast Brazil with widespread use in folk medicine. The major constituents of the essential oil of L. sidoides (EOLs) are thymol, p-cymene, myrcene, and caryophyllene. Several studies have shown that the EOLs and its constituents have pharmacological effects, including antibacterial, anti-inflammatory, antioxidant and neuroprotective activity. Therefore, this work aimed to investigate the effects of the EOLs and their main constituents on rat sciatic nerve excitability. The sciatic nerves of adult Wistar rats were dissected and mounted in a moist chamber. Nerves were stimulated by square wave pulses, with an amplitude of 40 V, duration of 100 µs to 0.2 Hz. Both EOLs and thymol inhibited compound action potential (CAP) in a concentration-dependent manner. Half maximal inhibitory concentration for CAP peak-to-peak amplitude blockade were 67.85 and 40 µg/mL for EOLs and thymol, respectively. CAP peak-to-peak amplitude was significantly reduced by concentrations ≥60 µg/mL for EOLs and ≥30 µg/mL for thymol. EOLs and thymol in the concentration of 60 µg/mL significantly increased chronaxie and rheobase. The conduction velocities of 1st and 2nd CAP components were also concentration-dependently reduced by EOLs and thymol in the range of 30-100 µg/mL. Differently from EOLs and thymol, p-cymene, myrcene and caryophyllene did not reduce CAP in the higher concentrations of 10 mM. These data demonstrated that EOLs and thymol inhibited neuronal excitability and were promising agents for the development of new drugs for therapeutic use.

Anethole improves the in vitro development of isolated caprine secondary follicles.

The aim of this study was to investigate the effect of three concentrations of anethole (30, 300, and 2000 µg/mL) on survival, antrum formation, follicular diameter, and oocyte maturation in the caprine species. The study also evaluated the effects of anethole on transcripts of ICAM-1, CAV-1, TIMP-2, and PAI-1 genes and levels of reactive oxygen species (ROS) in isolated goat preantral ovarian follicles before and after in vitro culture for 18 days. Preantral follicles were isolated from goat ovaries and individually cultured in alpha minimum essential medium modified (α-MEM+), defined as the control treatment, α-MEM+ supplemented with ascorbic acid at a concentration of 100 µg/mL (AA), or α-MEM+ supplemented with three different concentrations of anethole (30, 300, 2000 µg/mL) for a period of 18 days. Treatments were named as α-MEM+, AA, AN30, AN300, and AN2000, respectively. After culture, the follicles were opened, the cumulus oocytes complex (COCs) were removed and matured in vitro. The walls of the follicles were used for the quantitation of mRNA by quantitative real-time polymerase chain reaction. Finally, the medium collected at the end of culture was used for the measurements of ROS. After 18 days of culture, the AA treatment showed the percentage of intact follicles and follicular diameter significantly higher compared with the other treatments. However, daily growth rate, antrum formation, and also oocyte diameter were similar among the treatments. In addition, compared with AA, the rate of oocytes for in vitro maturation (diameter ≥ 110 µm) and the meiosis resumption rate were significantly higher in the treatments AN30 and AN2000, respectively. When assessing gene related to remodeling of the basement membrane, significant differences in mRNA levels for ICAM-1, CAV-1, TIMP-2, and PAI-1 were observed in comparison with Day 0, i.e., in the noncultured control. In addition, the ROS from Day 12, all treatments with the addition of anethole have significantly lower values of ROS than α-MEM+ and AA. In conclusion, the addition of anethole to the in vitro culture medium was able to improve the development of goat preantral follicles by reducing concentrations of ROS and increasing the percentage of oocytes able to resume meiosis.

Effects of endurance training on reduction of plasma glucose during high intensity constant and incremental speed tests in Wistar rats.

The aim of this research was to investigate the effects of endurance training on reduction of plasma glucose during high intensity constant and incremental speed tests in Wistar rats. We hypothesized that plasma glucose might be decreased in the exercised group during heavy (more intense) exercise. Twenty-four 10-week-old male Wistar rats were randomly assigned to sedentary and exercised groups. The prescription of endurance exercise training intensity was determined as 60% of the maximum intensity reached at the incremental speed test. The animals were trained by running on a motorized treadmill, five days/week for a total period of 67 weeks. Plasma glucose during the constant speed test in the exercised group at 20 m/min was reduced at the 14th, 21st and 28th min compared to the sedentary group, as well at 25 m/min at the 21st and 28th min. Plasma glucose during the incremental speed test was decreased in the exercised group at the moment of exhaustion (48th min) compared to the sedentary group (27th min). Endurance training positively modulates the mitochondrial activity and capacity of substrate oxidation in muscle and liver. Thus, in contrast to other studies on high load of exercise, the effects of endurance training on the decrease of plasma glucose during constant and incremental speed tests was significantly higher in exercised than in sedentary rats and associated with improved muscle and hepatic oxidative capacity, constituting an important non-pharmacological intervention tool for the prevention of insulin resistance, including type 2 diabetes mellitus.

The essential oil of Hyptis crenata Pohl ex Benth. presents an antiedematogenic effect in mice

Hyptis crenata, commonly known as "salva-do-Marajó", "hortelã-do-campo", and "hortelãzinha", is used in folk medicine in Northeast Brazil as tea or infusion to treat inflammatory diseases. Due to the pharmacological efficacy and the low toxicity of the essential oil of Hyptis crenata (EOHc), we decided to investigate the EOHc antiedematogenic effect in experimental models of inflammation. EOHc was administrated orally at doses of 10-300 mg/kg to male Swiss albino mice. Paw edema was induced by subcutaneous injection in the right hind paw of inflammatory stimuli (carrageenan, dextran, histamine, serotonin, and bradykinin) 60 min after administration of EOHc. EOHc significantly inhibited the induced edema. The inhibitory effect of EOHc on dextran-induced edema extended throughout the experimental time. For the 30, 100, and 300 mg/kg doses of EOHc, the inhibition was of 40.28±1.70, 51.18±2.69, and 59.24±2.13%, respectively. The EOHc inhibitory effect on carrageenan-induced edema started at 10 mg/kg at the second hour (h) and was maintained throughout the observation period. At 30, 100, and 300 mg/kg doses the inhibition started earlier, from 30 min. At the edema peak of 180 min, 56, 76, and 82% inhibition was observed for 30, 100, and 300 mg/kg doses, respectively. Additionally, the effect of EOHc on carrageenan-induced paw edema was influenced by the time of administration. The EOHc also inhibited myeloperoxidase activity. In conclusion, the EOHc showed a potent effect, both preventing and reversing the edema, consistent with its anti-inflammatory use in folk medicine.

Essential oil of Lippia alba and its main constituent citral block the excitability of rat sciatic nerves.

Lippia alba is empirically used for infusions, teas, macerates, and hydroalcoholic extracts because of its antispasmodic, analgesic, sedative, and anxiolytic effects. Citral is a mixture of trans-geranial and cis-neral and is the main constituent of L. alba essential oil and possesses analgesic, anxiolytic, anticonvulsant, and sedative effects. The present study evaluated the effects of the essential oil of L. alba (EOLa) and citral on compound action potentials (CAPs) in Wistar rat sciatic nerves. Both drugs inhibited CAP in a concentration-dependent manner. The calculated half-maximal inhibitory concentrations (IC50) of peak-to-peak amplitude were 53.2 µg/mL and 35.00 µg/mL (or 230 µM) for EOLa and citral, respectively. Peak-to-peak amplitude of the CAP was significantly reduced by 30 µg/mL EOLa and 10 µg/mL citral. EOLa and citral (at 60 and 30 µg/mL, values close to their respective IC50 for CAP blockade) significantly increased chronaxy and rheobase. The conduction velocity of the first and second CAP components was statistically reduced to ∼86% of control with 10 µg/mL EOLa and ∼90% of control with 3 µg/mL citral. This study showed that EOLa inhibited nerve excitability and this effect can be explained by the presence of citral in its composition. Both EOLa and citral showed inhibitory actions at lower concentrations compared with other essential oils and constituents with local anesthetic activity. In conclusion, these data demonstrate that EOLa and citral are promising agents in the development of new drugs with local anesthetic activity.

Selective inhibitory effects of niflumic acid on 5-HT-induced contraction of the rat isolated stomach fundus.

The effects of niflumic acid (NFA), an inhibitor of calcium-activated chloride currents I(Cl(Ca)), were compared with the actions of the voltage-dependent calcium channel (VDCC) blocker nifedipine on 5-hydroxtryptamine (5-HT)- and acetylcholine (ACh)-induced contractions of the rat isolated fundus. NFA (1 - 30 microM) elicited a concentration-dependent inhibition of contractions induced by 5-HT (10 microM) with a reduction to 15. 5+/-6.0% of the control value at 30 microM. 1 microM nifedipine reduced 5-HT-induced contraction to 15.2+/-4.9% of the control, an effect not greater in the additional presence of 30 microM NFA. In contrast, the contractile response to ACh (10 microM) was not inhibited by NFA in concentrations /=10 microM. Our results show that NFA can exert selective inhibitory effects on the chloride-dependent 5-HT-induced contractions of the rat fundus. The data support the hypothesis that activation of Cl((Ca)) channels leading to calcium entry via VDCCs is a mechanism utilized by 5-HT, but not by ACh, to elicit contraction of the rat fundus.

Comparative effects of niflumic acid and nifedipine on 5-hydroxytryptamine- and acetylcholine-induced contraction of the rat trachea.

The effects of niflumic acid, an inhibitor of Ca(2+)-activated Cl(-) (Cl((Ca))) channels, were compared with those of the voltage-dependent Ca(2+) channel (VDCC) blocker nifedipine on 5-hydroxytryptamine (5-HT)- and acetylcholine-induced contractions of the rat isolated trachea. Niflumic acid (3-100 microM) induced a concentration-dependent inhibition of 5-HT (10 microM)-induced contractions, with a reduction to 37.0+/-9.5% of the control at the highest concentration. One micromolar nifedipine, which completely blocked 60 mM KCl-induced contractions, reduced the response to 5-HT similarly to 39.2+/-11.5% of the control. The inhibition of the 5-HT response was not significantly different from that produced by the combined presence of nifedipine (1 microM) and niflumic acid (100 microM), suggesting that their effects were not additive. In contrast, neither niflumic acid (3-100 microM) nor nifedipine (1 microM) inhibited acetylcholine-induced contractions. The contraction to 5-HT (10 microM) in Cl(-)-free solution was decreased by more than approximately 85% of the control, whilst that of acetylcholine was reduced only by approximately 36%. Our data show that niflumic acid exerts selective inhibitory effects on 5-HT-induced contraction, and suggest that activation of Cl((Ca)) channels may be a mechanism whereby 5-HT (but not acetylcholine) induces Ca(2+) entry via VDCCs to elicit contraction.

Potassium-induced contractures in crab (Callinectes danae) muscle fibers.

The contractures induced by 20-200 mM [K+]o in single crab muscle fibers were resolved into two components. The first component, consisting of single twitches or brief tetanic contractions, was associated with electrogenic membrane responses. The second occurred after spiking subsided with an amplitude that increased linearly with the [K+]o between 20 and 90 mM. The amplitude and time course of the contractures elicited by a given [K+]o differed markedly between different fibers. Contracture reproducibility of a single fiber was best when 90 mM [K+]o was used. The K-induced contractures were abolished after brief (3 min) exposure of the fibers to a calcium-free solution and were greatly depressed by 8 mM procaine. The data suggest that the contractures require both Ca2+-influx across the sarcolemma and release of Ca2+ stored in the sarcoplasmic reticulum.

An optically coupled power stimulus isolation unit with high voltage and fast rise time output.

Recent technological developments have created new devices that could improve and simplify the construction of stimulus isolators. HEXFET transistors can switch large currents and hundreds of volts in nanoseconds. The newer opto-isolators can give a pulse rise time of a few nanoseconds, with output compatible with MOSFET devices, in which delays are reduced to nanoseconds. Integrated DC/DC converters are now available. Using these new resources we developed a new electrical stimulus isolator circuit with selectable constant-current and constant-voltage modes, which are precise and easy to construct. The circuit works like a regulated power supply in both modes with output switched to zero or to free mode through an opto-isolator device. The isolator analyses showed good practical performance. The output to ground resistance was 10(11) ohms and capacitance 35 picofarads. The rise time and fall time were identical (5 microseconds) and constant. The selectable voltage or current output mode made it very convenient to use. The current mode, with higher output resistance values in low current ranges, permits intracellular stimulation even with tip resistances close to 100 megaohms. The high compliance of 200 V guarantees the value of the current stimulus. The very low output resistance in the voltage mode made the device highly suitable for extracellular stimulation with low impedance electrodes. Most importantly, these characteristics were achieved with a circuit that was easy to build and modify and assembled with components available in Brazil.

Effects of dichlorobenzamil, a sodium-calcium exchange inhibitor, on the calcium paradox and the sodium withdrawal contractures of frog atrial muscle.

1. The effects of dichlorobenzamil (DCB), an amiloride derivative and potent inhibitor of Na-Ca exchange in cardiac sarcolemmal vesicles and isolated cardiac myocytes, were investigated in two paradigms involving Na-Ca exchange, namely the Ca2+ paradox and the Na+-withdrawal contractures of frog atrial muscle strips. 2. Pretreatment with DCB (10-100 microM) inhibited in a dose-dependent manner the contractures elicited by reexposure of the atrial strips to the control Ringer solution after a 5-20 min equilibration with a Ca2+-free saline (Ca2+-readmission contractures; Ca2+ paradox). These contractures were not inhibited, however, when DCB was applied after the preparation had been exposed to the Ca2+-free saline, but before the reexposure to the control Ringer solution. 3. DCB (10-100 microM) did not inhibit the contractures elicited by Na+-deficient saline (Na+-withdrawal contractures) in atrial strips pretreated or not with acetylstrophantydin. This result suggests that, under our experimental conditions, DCB failed to substantially inhibit the Ca2+ influx mediated by Na-Ca exchange. 4. The duration of the plateau of the action potentials of atrial cells equilibrated with Ca2+-free saline was reduced from 1.42 +/- 0.27 s to 0.61 +/- 0.13 s by 50 microM DCB (P less than 0.001). This was attributed to blockade of Na+ currents through modified L-type Ca2+ channels. 5. It is proposed that the shortening of the Na+-dependent action potentials can account for the inhibition of the Ca2+-readmission contractures, because these contractures have a steep dependence on the Na+ influx and intracellular Na+ accumulation that occurs during the Ca2+-free period. 6. The results of this study support the conclusion that DCB has multiple effects on heart muscle, including a potent blockade of Ca2+ channels, and its use as a selective inhibitor of Na-Ca exchange in cellular systems is unwarranted.

Role of mast cell- and non-mast cell-derived inflammatory mediators in immunologic induction of synaptic plasticity.

We have previously discovered a long-lasting enhancement of synaptic transmission in mammal autonomic ganglia caused by immunological activation of ganglionic mast cells. Subsequent to mast cell activation, lipid and peptide mediators are released which may modulate synaptic function. In this study we determined whether some mast cell-derived mediators, prostaglandin D2 (PGD2; 1.0 microM), platelet aggregating factor (PAF; 0.3 microM) and U44619 (a thromboxane analogue; 1.0 microM), and also endothelin-1 (ET-1; 0.5 microM) induce synaptic potentiation in the guinea pig superior cervical ganglion (SCG), and compared their effects on synaptic transmission with those induced by a sensitizing antigen, ovalbumin (OVA; 10 micrograms/ml). The experiments were carried out on SCGs isolated from adult male guinea pigs (200-250 g) actively sensitized to OVA, maintained in oxygenated Locke solution at 37 degrees C. Synaptic potentiation was measured through alterations of the integral of the post-ganglionic compound action potential (CAP). All agents tested caused long-term (LTP; duration > or = 30 min) or short-term (STP; < 30 min) potentiation of synaptic efficacy, as measured by the increase in the integral of the post-ganglionic CAP. The magnitude of mediator-induced potentiation was never the same as the antigen-induced long-term potentiation (A-LTP). The agent that best mimicked the antigen was PGD2, which induced a 75% increase in CAP integral for LTP (antigen: 94%) and a 34% increase for STP (antigen: 91%). PAF-, U44619-, and ET-1-induced increases in CAP integral ranged for LTP from 34 to 47%, and for STP from 0 to 26%. These results suggest that the agents investigated may participate in the induction of A-LTP.

Technical problems related to the analysis of the effects of inorganic phosphate on cardiac muscle.

1. We describe how potential artifacts (due to solution composition, buffering capacity of the bathing medium, size of the skinned fiber preparation, permeability of the sarcoplasmic reticulum (SR) vesicles, and proper Kd for Ca2+ of the fluorescent indicator used to measure Ca2+ transport can be avoided in order to determine the effects of inorganic phosphate (Pi, or any other ion) on maximum Ca2+ activated force (Fmax) and Ca2+ sensitivity of skinned cardiac muscle fibers, and Ca(2+)-ATPase activity and uptake properties of isolated cardiac SR-enriched vesicles. 2. To maintain the ionic strength of the bathing medium constant when adding Pi, other ions must be removed. We found that because some salts have a depressant effect on Fmax independent of increased ionic strength (e.g. KCl) while others do not (e.g. Na-acetate), the salt used to adjust ionic strength influences the measured depressant effect of Pi on Fmax. 3. The sensitivity to Ca2+ of the contractile apparatus depends on the sum of the [Na+] and [K+] in the bathing medium. However, we found that the effect of Pi on Ca2+ sensitivity was not significantly influenced by the small changes in the sum of [Na+] and [K+] that were associated with the addition of Pi. 4. The skinned fiber preparations were approximately cylindrical bundles with diameters ranging between 100 and 250 microns. We found that the effect of Pi on Fmax was not influenced by diffusion limitations over this range of bundle diameters. 5. The pH buffering capacity of the bathing solution affects Fmax at pH 6.6. We found that the buffering effect of Pi can influence the mechanical response of skinned fibers independent of a direct effect of Pi on the contractile apparatus when the buffering capacity of the control solution is low. 6. When the Ca(2+)-ATPase of isolated SR vesicles is activated by Ca2+ and MgATP, the vesicles accumulate Ca2+. Unless the vesicles are permeabilized with a Ca2+ ionophore (ionomycin) and the pH adequately buffered, maximum ATPase activity will be underestimated, the broadness of the curve relating Ca2+ to Ca(2+)-ATPase rate overestimated, and the sensitivity to Pi overestimated. 7. The ionic milieu of isolated SR vesicles changes the apparatus dissociation constant (Kd) of Ca(2+)-Fura-2, a fluorescent dye used to quantify SR Ca2+ transport rates. In order to accurately measure the inhibitory effect of Pi on Ca2+ uptake, the influence of Pi on the Kd of fura-2 for Ca2+ must be taken into account.