Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.

Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.

The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.

Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.

Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).

These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.     

Role of calcium in the bombesin-induced intestinal CCK release in rats

In the isolated vascularly perfused rat duodenojejunum, vascular infusion of bombesin (100 nM) provoked an early, transient (6 min) release of CCK (500% of basal), followed by a sustained response (400% of basal). The calcium chelator EGTA (2 mM) reduced the early peak and abolished the second phase of CCK release. A similar variation was evoked by verapamil (10 μM), whereas diltiazem (100 μM), nifedipine (50 μM), and ω-conotoxin (100 nM) had no significant effect. It is concluded that bombesin-induced CCK release from rat intestine is dependent on the availability of extracellular calcium and on the activation of calcium channels sensitive to blockers of the phenylalkylamine family.     

Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: involvement of mechanical stress-activated calcium influx, PKC and Src activation

There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca²⁺]_i) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 μM) increased [Ca²⁺]_i by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y₂ receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5 mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 μM), a protein kinase C (PKC) inhibitor, and PP1 (50 μM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca²⁺-free extracellular medium (containing 0.5 mM EGTA) and the use of gadolinium (5 μM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y₂ receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.     

Cholinesterase inhibitory and spasmolytic potential of steroidal alkaloids

A new steroidal alkaloid, isosarcodine (1) along with four known bases, sarcorine (2), sarcodine (3), sarcocine (4) and alkaloid-C (5) were isolated from the MeOH extract of Sarcococca saligna. The structures of these alkaloids were identified by spectral data interpretation. These compounds were subjected to acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition studies, and were found to be noncompetitive inhibitors of AChE (K_i = 21.8, 90.3, 32.2, 16.0 and 50.0 μM, respectively) and uncompetitive or noncompetitive inhibitors of BChE (K_i = 8.3, 7.5, 15.6, 5.0 and 12.0 μM, respectively).

The compounds (2–5) also showed dose-dependent spasmolytic activity in the rabbit jejunum intestinal preparations and also relaxed the high K⁺ (80 mM)-induced contraction, indicative of a calcium channel-blocking mechanism.

Structure–activity relationship suggested that the nitrogen substituents at C-3 and/or C-20 of steroidal skeleton and the hydrophobic properties of the pregnane skeleton are the key structural features contributed to the inhibitory potency of these steroidal alkaloids against AChE and BChE.     

Binding and biologic activity of neurotensin in guinea pig ileum

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38°C. NT contracted the longitudinal muscle (ED₅₀, 0.3 nM), the 2–3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 μM), ≥50% of the response was blocked and the residual effect gave an ED₅₀ of 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 μM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED₅₀, 0.3 nM) and one substance P (ED₅₀, 1.4 nM). Using membrane preparations and ¹²⁵I-labeled NT, specific, high affinty receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (K_ds: 0.1 nM and 2 nM). Sodiu ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg⁺⁺ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.     

Opposite modulation of capsaicin-evoked substance P release by glutamate receptors

Substance P and glutamate are present in primary afferent C-fibers and play important roles in persistent inflammatory and neuropathic pain. In the present study, we have examined whether activation of different glutamate receptor subtypes modulates the release of substance P evoked by the C-fiber selective stimulant capsaicin (1 μM) from rat trigeminal nucleus slices. The selective NMDA glutamate receptor agonist L-CCG-IV (1–10 μM) enhanced capsaicin-evoked substance P release about 100%. This facilitatory effect was blocked by 0.3 μM MK-801, a selective NMDA receptor antagonist. The metabotropic glutamate receptor agonists L-AP4 (group III) and DHPG (group I) (30–100 μM) inhibited capsaicin-evoked substance P release by approximately 60%. These inhibitory effects were blocked by the selective metabotropic glutamate receptor antagonist (±)-MCPG (5 μM). On the other hand, AMPA and kainate (0.1–10 μM), did not significantly affect capsaicin-evoked substance P release. Thus, substance P release from non-myelinated primary afferents, and possibly nociception, may be under the functional antagonistic control of some metabotropic and ionotropic glutamate receptor subtypes.     

Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines

Chalcones xanthohumol (X) and desmethylxanthohumol (DMX), present in hops (Humulus lupulus L.), and the corresponding flavanones isoxanthohumol (IX, from X), 8-prenylnaringenin (8-PN, from DMX), and 6-prenylnaringenin (6-PN, from DMX), have been examined in vitro for their anti-proliferative activity on human prostate cancer cells PC-3 and DU145. X proved to be the most active compound in inhibiting the growth of the cell lines with IC₅₀ values of 12.3±1.1 μM for DU145 and 13.2±1.1 μM for PC-3. 6-PN was the second most active growth inhibitor, particularly in PC-3 cells (IC₅₀ of 18.4±1.2 μM). 8-PN, a highly potent phytoestrogen, exhibited pronounced anti-proliferative effects on PC-3 and DU145 (IC₅₀ of 33.5±1.0 and 43.1±1.2 μM, respectively), and IX gave comparable activities (IC₅₀ of 45.2±1.1 μM for PC-3 and 47.4±1.1 μM for DU145). DMX was the least active compound. It was evidenced for the first time that this family of prenylated flavonoids from hops effectively inhibits proliferation of prostate cancer cells in vitro.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D₄ receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 μM), and by arachidonic acid (AA), melittin and LTD₄. FPL55712 (0.1 and 1 μM) inhibited contractions induced by AA (100 μM) and the phospholipase A₂ activator melittin (3 μg/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 μM) was a more effective inhibitor of the melittin response than the response. FPL55712 inhibited contractions induced by OA (100 μg/ml) more than by A23187 (1 μg/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC₄ conversion to LTD₄. NDGA (10 μM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 μM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD₄-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD₄-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.     

Dithioerythritol (DTE) prevents inhibitory effects of triphenyltin (TPT) on the key enzymes of the human sex steroid hormone metabolism

Organotins are known to induce imposex (pseudohermaphroditism) in marine neogastropods and are suggested to act as specific endocrine disruptors, inhibiting the enzyme-mediated conversion of steroid hormones. Therefore, we investigated the in vitro effects of triphenyltin (TPT) on human 5-reductase type 2 (5-Re 2), cytochrome P450 aromatase (P450arom), 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD 3), 3β-HSD type 2 and 17β-HSD type 1 activity. First, the present study demonstrates that significant amounts of TPT occurred in the blood of eight human volunteers (0.17–0.67 μg organotin cation/l, i.e. 0.49–1.92 nmol cation/l). Second, TPT showed variable inhibitory effects on all the enzymes investigated. The mean IC₅₀ values were 0.95 μM for 5-Re 2 (mean of n=4 experiments), 1.5 μM for P450arom (n=5), 4.0 μM for 3β-HSD 2 (n=1), 4.2 μM for 17β-HSD 3 (n=3) and 10.5 μM for 17β-HSD 1 (n=3). To exclude the possibility that the impacts of TPT are mediated by oxidizing essential thiol residues of the enzymes, the putative compensatory effects of the reducing agent dithioerythritol (DTE) were investigated. Co-incubation with DTE (n=3) resulted in dose-response prevention of the inhibitory effects of 100 μM deleterious TPT concentrations on 17β-HSD 3 (EC₅₀ value of 12.9 mM; mean of n=3 experiments), 3β-HSD 2 (0.90 mM; n=3), P450arom (0.91 mM; n=3) and 17β-HSD 1 (0.21 mM; n=3) activity. With these enzymes, the use of 10 mM DTE resulted in an at least 80% antagonistic effect, whereas, the effect of TPT on 5-Re 2 was not compensated. In conclusion, the present study shows that TPT acts as an unspecific, but significant inhibitor of human sex steroid hormone metabolism and suggests that the inhibitory effects are mediated by the interaction of TPT with critical cysteine residues of the enzymes.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

Dynamic properties of stretch-activated K+ channels in adult rat atrial myocytes

The effect of mechanical stress on the heart's electrical activity has been termed mechanoelectric feedback. The response to stretch depends upon the magnitude and the waveform of the stimulus, and upon the timing relative to the cardiac cycle. Stretch-activated ion channels (SACs) have been regarded as the most likely candidates for serving as the primary transducers of mechanical stress. We explored the steady state and dynamic responses of single channels in adult rat atrial cells using the patch clamp with a pressure clamp. Surprisingly, we only observed K⁺-selective SACs, probably of the 2P domain family. The channels were weakly outward rectifying with flickery bursts. In cell attached mode, the mean conductance was 74±14 and 65±16 pS for +60 and −60 mV, respectively (140 mM [K⁺]_out, 2 mM [Mg²⁺]_out and 0 mM [Ca²⁺]_out). The latency of the response to pressure steps was 50–100 ms and the time to peak 400 ms. About half of the channels in cell-attached patches showed adaptation/inactivation where channel activity declined to a plateau of 20–30% of peak in 1 s. The time dependent behavior of these SACs is generally consistent with whole-cell currents observed in chick and rat ventricular cells, although the net current was outward rather than inward.     

Role of calcium in the potentiating effect of phorbol ester on KCl-induced vasocontraction

The mechanism of the potentiating effect of phorbol ester on potassium-induced contraction in rat aorta was investigated. The contractile response to KCl in the medium containing 0.5 mM CaCl2 was significantly increased by pretreatment with 10(-8) M phorbol 12-myristate 13-acetate (PMA), but not with 10(-7) M 4 alpha-phorbol. The dose-response curve to calcium in 30 mM KCl-induced contraction was shifted to the left by PMA pretreatment and the EC50 value (the concentration producing a half maximal response) of calcium was significantly lower in aorta pretreated with PMA than in the control. On the other hand, calcium influx stimulated by 30 mM KCl was not changed by PMA pretreatment. Both the contractile response and the corresponding calcium influx induced by 30 mM KCl were abolished by preincubation with 10(-6) M verapamil for 45 min. These results suggest that activation of protein kinase C potentiates the contractile response to KCl by increasing the sensitivity of the intracellular contractile apparatus for calcium.     

An interaction between benzodiazepines and neuroactive steroids at GABA A receptors in cultured hippocampal neurons

Neurosteroids are modulators of several receptors and ion channels and are implicated in the pathophysiology of several neuropsychiatric diseases including hepatic encephalopathy (HE). The neurosteroid, allopregnanolone, a positive allosteric modulator of GABA_A receptors, accumulates in the brains of HE patients where it can potentiate GABA_A receptor-mediated responses. Attenuation of the effects of neurosteroids on GABA-ergic neurotransmission is therefore of interest for the management of HE. In the present study, we determined the effect of the benzodiazepine partial inverse agonist, Ro15-4513, and the benzodiazepine antagonist, flumazenil on modulation of the GABA_A mediated chloride currents by allopregnanolone and on spontaneous synaptic activity in cultured hippocampal neurons using the patch-clamp technique. Allopregnanolone (0.03–0.3 μM), dose-dependently potentiated GABA-induced currents, an action significantly reduced by Ro15-4513 (10 μM). In contrast, flumazenil (10 μM) had no effect on the ability of allopregnanolone to potentiate GABA_A currents but it blocked the effects of Ro15-4513. The frequency of spontaneous synaptic activity was significantly reduced in the presence of allopregnanolone (0.1 μM) from 1.5 ± 0.7 to 0.1 ± 0.04 Hz. This action was partially reversed by Ro15-4513 (10 μM) but was not significantly influenced by flumazenil (10 μM). These findings suggest that the beneficial affects of Ro15-4513 in experimental HE result from attenuation of the effects of neurosteroids at GABA_A receptors. Our results may provide a rational basis for the use of benzodiazepine inverse agonists in the management and treatment of hepatic encephalopathy in patients with liver failure.     

Opioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo

Capsaicin has been shown to evoke the release of substance P (SP) from small diameter primary afferent fibers. Using an in vivo perfusion of the rat spinal cord, this study examined the pharmacology of opioid receptor systems which modulate the capsaicin-evoked release of SP. The addition of capsaicin (200 μM) to the perfusate raised SP-like immunoreactivity (SP-LI) from resting levels of 31±5 to 74±14 pg/ml or an increase of 139% above the baseline. Using high pressure liquid chromatography (HPLC) the identity of the released SP-LI was determined to coelute primarily with authentic SP or the oxidized form of SP. Opioid receptor agonists were added to the perfusate and their ability to inhibit capsaicin-evoked release of SP-LI was assessed. Morphine (10–100 μM), DAGO (1–100 μM), DPLPE (10–100 μM), but not U50488H (100 μM) produced a dose-dependent reduction in the capsaicin-evoked release of SP-LI. Pretreatment with the opioid receptor antagonist naloxone (1 mg/kg, IP) had no effect on the basal or capsaicin-evoked release of SP-LI. Naloxone pretreatment was able to antagonize completely the opioid-produced inhibition of capsaicin-evoked SP-LI release. These data indicate that the release of SP from primary afferent fibers can be modulated by the activation of mu or delta but not kappa opioid receptors. Further, these data support the hypothesis that spinally administered mu and delta opioid agonists may produce their antinociceptive effect through the presynaptic inhibition of neuropeptide release from small diameter primary afferent fibers.     

Effects of calcium binding on the structure and stability of human growth hormone

Thermodynamic analysis of calcium ions binding to human growth hormone (hGH) was done at 27 °C in NaCl solution, 50 mM, using different techniques. The binding isotherm for hGH-Ca²⁺ was obtained by two techniques of ionmetry, using a Ca²⁺-selective membrane electrode, and isothermal titration calorimetry. Results obtained by two ionmetric and calorimetric methods are in good agreement. There is a set of three identical and non-interacting binding sites for calcium ions. The intrinsic dissociation equilibrium constant and the molar enthalpy of binding are 52 μM and −17.4 kJ/mol, respectively. Temperature scanning UV–vis spectroscopy was applied to elucidate the effect of Ca²⁺ binding on the protein stability, and circular dichroism (CD) spectroscopy was used to show the structural change of hGH due to the metal ion interaction. Calcium ions binding increase the protein thermal stability by increasing of the alpha helix content as well as decreasing of both beta and random coil structures.     

Electrophysiological properties and gap junction coupling of striatal astrocytes

The striatum is the biggest nucleus of the basal ganglia and receives input from almost all cortical regions, substantia nigra and the thalamus. Striatal neuronal circuitry is well characterized, but less is known about glial physiology. To this end, we evaluated astrocyte electrophysiological properties using whole-cell patch-clamp recording in dorsal striatal brain slices from P15 to P21 rat. The majority of cells (95%) were passive astrocytes that do not express any detectable voltage-gated channels. Passive astrocytes were subcategorized into three groups based on time-dependent current properties. The observed proportion of the different astrocyte subtypes did not change within the age range evaluated here, but was modulated during reduction of specific conductances and gap junction coupling. Striatal astrocytes were extensively interconnected and closure of gap junctions with octanol (1 mM), carbenoxolone (100 μM) or increased intracellular calcium (2 mM), significantly altered intrinsic properties. When simultaneously blocking potassium channels and gap junction coupling almost no passive conductance was detected, implying that the major currents in striatal astrocytes derive from potassium and gap junction conductance. Uncoupling of the syncytium reduced currents activated in response to a hyperpolarizing pulse, suggesting that changes in gap junction coupling alters astrocyte electrophysiological responses. Our findings indicate that the prevalent gap junction coupling is vital for astrocyte function in the striatum, and that whole-cell recordings will be distorted by currents activated in neighboring cells.     

Substance P contracts the human iris sphincter: possible modulation by endogenous enkephalinase.

Substance P-immunoreactive neurons have been found in the irides of many species including humans. In several species, substance P has been shown to induce contraction of the sphincter muscle but this action of substance P has not been previously demonstrated in the human eye. Using an eye cup model in which the sensitivity of the iris muscle to substance P is increased compared to the isolated sphincter muscle, we have observed that nanomolar amounts of substance P induced contraction of the sphincter in the human iris. This contractile response was enhanced in eyes pretreated with thiorphan, an enkephalinase inhibitor, suggesting that endogenous enkephalinase (E.C. 3.4.24.11) may modulate the substance P contraction in the human iris. Further support for this hypothesis was the finding of enkephalinase-like immunoreactivity and enzyme activity in the human iris sphincter muscle.