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.     

Time and concentration dependency of the toxicity of excitatory amino acids on cerebral neurones in primary culture

The cytotoxicity of the glutamate receptor agonists, N-methyl- -aspartate (NMDA), kainate (KA) and (RS)--amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) on cultured cerebral cortex neurones was monitored as a function of exposure time and concentration by following the release into the culture medium of the cytoplasmic enzyme lactate dehydrogenase from the neurones. Chronic exposure of the cells to different concentrations of the agonists showed that AMPA was the most potent excitotoxin (ED₅₀ 10 μM) followed in potency by NMDA (ED₅₀ 65 μM) and KA (ED₅₀ 100 μM). Experiments in which the neurones were exposed for different periods of time to fixed concentrations of the agonists showed that after short exposure times (1–3 min) cells survived for more than 24 h after removal of the agonists but after longer exposure times (5–10 min) cells survived for time periods ranging from 25 min to 6 h depending upon the exposure time and the nature of the agonist. The results of the latter experiments indicate that even short exposure times trigger processes in the cell membranes which even after removal of the excitotoxin will lead to neuronal death.     

The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats

Previous studies suggested that electroacupuncture (EA) can suppress opioid dependence by the release of endogenous opioid peptides. To explore the site of action and the receptors involved, we tried to inject highly specific agonists for μ-, δ- and κ-opioid receptors into the CNS to test whether it can suppress morphine-induced conditioned place preference (CPP) in the rat. Male Sprague–Dawley rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP model. This CPP can be prevented by (a) i.p. injection of 3 mg/kg dose of morphine, (b) intracerebroventricular (i.c.v.) injection of micrograms doses of the selective μ-opioid receptor agonist DAMGO, δ-agonist DPDPE or κ-agonist U-50,488H or (c) microinjection of DAMGO, DPDPE or U50488H into the shell of the nucleus accumbens (NAc). The results suggest that the release of endogenous μ-, δ- and κ-opioid agonists in the NAc shell may play a role for EA suppression of opiate addiction.     

Kainate-induced stimulation of amino acid release from primary astroglial cultures of the rat hippocampus

The effects of the excitatory amino acid analogs kainate (KA) and N-methyl- -aspartate (NMDA) on release of amino acids from astrocytes in primary culture were investigated. Under basal conditions, glutamine was present in the medium at 15 μM. The levels of serine and taurine were 1.5 and 2.0 μM, respectively, while the concentration of other amino acids was below 1 μM. At 10 μM, KA did not affect amino acid release, whereas 100 μM KA enhanced glutamine release by 34% and taurine release by 85%. At 1 mM, KA stimulated the release of all amino acids measured. However, while most amino acids increased by 50–150%, glutamate and aspartate were elevated by more than 3000%. The effect of KA was greatly reduced by 1 mM kynurenate, an excitatory amino acid receptor antagonist. 1 mM NMDA did not stimulate amino acid release from the cultures. The results indicate that astrocytes are endowed with KA-receptive sites, but they do not seem to possess NMDA receptors.     

The regulation of steroidogenesis by opioid peptides in porcine theca cells

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.     

Strychnine-insensitive glycine receptors in embryonic chick retina: characteristics and modulation of NMDA neurotoxicity

In the mammalian brain, the (NMDA) subtype of glutamate receptor is coupled to a cation channel and a strychnine-insensitive glycine receptor. The present paper demonstrates the presence of NMDA receptor-coupled strychnine-insensitive glycine receptors in embryonic chick retina. Both glycine and 1-aminocyclopropanecarboxylic acid (ACPC) exhibited similar potencies (271 ± 39 vs 247 ± 39 nM, respectively) as inhibitors of strychnine-insensitive [³H]glycine binding to retinal membranes. Moreover, glycine and ACPC enhanced [³H]MK-801 binding to sites within the NMDA-coupled cation channel in retinal membranes with potencies comparable to those reported in rat brain. While the potency of ACPC was significantly higher than glycine (EC₅₀ 54±12 vs 256±57 nM, P < 0.02) in this measure, there were no significant differences in the maximum enhancement (efficacy) of [³H]MK-801 binding by these compounds. Since glycine appears to be required for the operation of NMDA-coupled cation channels, we examined the effects of glycine and ACPC on NMDA-induced acute excitotoxicity in the 14-day embryonic chick retina. Histological evaluation of retina revealed that either ACPC (10–100 μM) or glycine (200 μM) attenuated NMDA- induced (200 μM) retinal damage, and a combination of these agents produced an enhanced protection against acute NMDA toxicity. ACPC (100 μM), but not MK-801 (1 μM) also afforded a modest protection against kainate-induced (25 μM) retinal damage. These findings demonstrate that while strychnine-insensitive glycine receptors are present in embryonic chick retina, occupation of these sites does not augment the cytotoxic actions of NMDA. Moreover, the ability of ACPC and glycine to attenuate NMDA-induced cytotoxicity does not appear to be mediated through occupation of these sites.     

Effects of cholecystokinin peptides and neurotensin on dopamine release and metabolism in the rostral and caudal part of the nucleus accumbens using intracerebral dialysis in the anaesthetized rat

By means of intracerebral microdialysis effects of cholecystokinin peptides and neurotensin administered via the microdialysis probe have been studied on dopamine release and metabolism in the nucleus accumbens and neostriatum of the halothane anaesthetized male rat. Levels of extra cellular dopamine (DA) and its metabolites 3,4 dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assessed in nuc. accumbens (rostral and caudal part) using high performance liquid chromatography in combination with electrochemical detection.

(1) In the rostral part of the nuc. accumbens CCK-8 (10 and 100 μM), CCK-33 (100 μM) but not CCK-4 (10 and 100 μM) increased the levels of DA in the perfusate without increasing the extracellular levels of DOPAC and HVA. (2) In the caudal nuc. accumbens CCK-8 and CCK-4 in concentrations of 10 μM and 100 μ M of CCK-33 had no effect on DA release and metabolism, since the extracellular levels of DA, DOPAC and HVA were not changed. (3) In the rostral nuc. accumbens perfusion with 10 μM of neurotensin but not with any other concentration of neurotensin (0.01, 0.1, 1 and 100 μM) increased the levels of DA in the extracellular fluid. (4) In the caudal nuc. accumbens a 40 min perfusion with neutrotensin produced a concentration dependent increase of the levels of DA in the perfusate (peak action at 10 μ M) which in this case was associated with increases in the extracellular levels of DOPAC and HVA. (5) By means of receptor autoradiography using (3-[¹²⁵I]iodotyrosyl³) neurotensin it was found that a 40 min perfusion with this radioligand in the rostral nuc. accumbens reached a total volume of 0.051 mm³. The diffusion of the radioligand was limited to the rostral or caudal part of the nuc. accumbens depending upon the site of placement of the dialysis probe.

The results indicate the existence of cholecystokinin (CCK) receptors in the rostral nuc. accumbens, which are sensitive to CCK-8 and CCK-33 but not to CCK-4, and which facilitate DA release without producing any detectable increase in DA metabolites. In contrast, such receptors do not appear to play a similar role in the regulation of DA release in the caudal nuc. accumbens, where DA terminals contain CCK-like immunoreactivity. Furthermore, the results indicate that neurotensin receptors exist both in the rostral and caudal nuc. accumbens, where they inter alia enhance the release of DA. In the caudal nuc. accumbens these effects of neurotensin are also associated with an increase of DA metabolites, possibly suggesting that in this region neurotensin receptors may also control DA synthesis.     

Effect of Chronic Administration of Morphine, U-50, 488H and [D-Pen, D-Pen]enkephalin on the Concentration of cGMP in Brain Regions and Spinal Cord of the Mouse

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of morphine, U-50,488H and [ -Pen², -Pen⁵]enkephalin on the concentration of cGMP in brain regions and spinal cord of the mouse. Peptides 18(10) 1629–1634, 1997.—The effects of chronic administration and subsequent withdrawal of μ-, κ- and δ-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at μ-, κ- and δ-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla(76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of μ- and κ- opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to μ- and κ- but not to δ-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by μ-, κ- and δ-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to μ-, κ- and δ-opioid receptor agonists.     

Opposite Effects of δ and μ Opioid Receptor Agonists on the In Vitro Release of Substance P-Like Material from the Rat Spinal Cord

Superfusion of slices from the dorsal half of the lumbar enlargement of rat spinal cord with Krebs-Henseleit medium supplemented with 30 microM bacitracin allowed the collection of substance P-like immunoreactive material (SPLI), which was released at a rate of approximately 10 pg/4 min. Tissue depolarization by an excess of K+ (30-60 mM) or veratridine (50 microM) induced a marked increase in SPLI outflow, provided that Ca2+ was present in the superfusing fluid. K+- or veratridine-induced SPLI overflow could be modulated in opposite directions by mu and delta opioid receptor agonists. Thus, the two preferential mu agonists Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO; 10 microM) and Tyr-D-Ala-Gly-MePhe-Met(O)5-OH (FK-33824; 0.1 microM) enhanced SPLI overflow from depolarized tissues, whereas the selective delta agonists Tyr-D-Thr-Gly-Phe-Leu-Thr (deltakephalin; 3 microM) and [2-D-penicillamine, 5-D-penicillamine]enkephalin (50 microM) reduced it. The effect of DAGO was antagonized by a low concentration (1 microM) of naloxone but not by the selective delta antagonist ICI-154129 (50 microM). In contrast, the latter drug prevented the inhibitory influence of delta agonists on K+-induced SPLI release. Complementary experiments with morphine (10 microM) and [2-D-alanine, 5-D-leucine]enkephalinamide (3 microM), in combination with 1 microM naloxone or 50 microM ICI-154129 for the selective blockade of mu or delta receptors, respectively, confirmed that the stimulation of mu receptors increased, whereas the stimulation of delta receptors reduced, SPLI overflow. The results suggest that, at the spinal level, and antinociceptive action of delta but not mu agonists might involve a presynaptic inhibition of substance P-containing primary afferent fibers.     

Solubilization of active GLP-1 (7–36)amide receptors from RINm5F plasma membranes

Glucagon-like peptide-1 (7–36)amide (GLP-1 (7–36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7–36)amide have been described in RINm5F cells. We have solubilized active GLP-1 (7–36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-β-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7–36)amide to the solubilized receptor was inhibited conentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K_a= 0.26 ± 0.03 nM and B_max= 65.4 ± 21.24 fmol/mg of protein for the membrane-bound receptor and K_a= 22.54 ± 4.42 μM and B_max= 3.9 ± 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-γ-S nor GDP-β-S affected binding or labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7–36)amide receptor in a functional state and opens up the possibility for further molecular characterization of the receptor protein.     

Capsaicin-evoked bradycardia in anesthetized guinea pigs is mediated by endogenous tachykinins

The present study was done to characterize the effects of endogenous tachykinins on heart rate in urethane-anesthetized guinea pigs. Intravenous injection of capsaicin (32 nmol/kg) was used to evoke release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac sensory nerve fibers. Such injections caused a brief decrease in heart rate (− 37 ± 7 beats/min, n = 6) that was followed by a more prolonged increase (+ 44 ± 10 beats/min). Blood pressure was lowered by − 11 ± 2 mmHg. Bilateral vagotomy did not affect the chronotropic or depressor responses to capsaicin, but atropine (1 µmol/kg) nearly abolished the bradycardic response (− 8 ± 3 beats/min, n = 7). Combined blockade of NK₂ and NK₃ receptors, with SR48968 and SR14801 respectively, also caused a significant reduction of capsaicin-evoked bradycardia (− 14 ± 3 beats/min, n = 4) but did not affect bradycardia evoked by vagal nerve stimulation. Blockade of CGRP receptors eliminated capsaicin-evoked tachycardia and prolonged the capsaicin-evoked bradycardia. These findings suggest that capsaicin-evoked bradycardia in the anesthetized guinea pig is mediated by tachykinins that stimulate cardiac cholinergic neurons. This effect appears to be truncated by the positive chronotropic action of CGRP that is also released from cardiac afferents by capsaicin.     

μ Receptors and opioid cardiovascular effects in the NTS of rat

In order to assess the potential role of mu (μ) and delta (δ) opiate receptors in the central regulation of the cardiovascular and respiratory systems, the cardiovascular and respiratory effects of the relatively selective μ-opioid agonist D-Ala², MePhe⁴, Gly-ol⁵ enkephalin (DAGO) and relatively selective δ-agonist D-Ala²-D-Leu⁵ enkephalin (DADL) were compared following microinjection of these compounds into the nucleus tractus solitarius of pentobarbital-anesthetized rats. Both opioid agonists produced dose dependent increases in systolic and diastolic blood pressure as well as heart rate; but DAGO was nearly ten times more potent in eliciting these changes. Respiratory rate was increased by DADL and by lower doses of DAGO, but was depressed by higher doses of DAGO. Tidal volume was depressed by both peptides. These data support the concept that the cardiovascular pressor responses and tachycardia as well as the respiratory effects of opioids in the rat NTS are mediated by μ receptors.     

Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme

In addition to plasma metabolism of substance P (SP) by angiotensin converting enzyme (ACE; EC 3.4.15.1) (<1.0 nmol/min/ml), the majority of SP hydrolysis by rat and human plasma was due to dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (3.15–5.91 nmol/min/ml), which sequentially converted SP to SP(3–11) and SP(5–11). In turn, the SP(5–11) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidase M (AmM; EC 3.4.11.2) (24.2–25.5 nmol/min/ml). The K_m values of SP for DAP IV and of SP(5–11) for AmM ranged from 32.7 to 123 μM. In contrast, neurokinin A (NKA) was resistant to both ACE and DAP IV but was subject to N-terminal hydrolysis by AmM (3.76–10.8 nmol/min/ml; K_m=90.7 μM. These data demonstrate differential processing of SP and NKA by specific peptidases in rat and human plasma.     

High-affinity carbamate analogues of morphinan at opioid receptors

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.     

Effect of sole and combined pre-treatments on reserve accumulation, survival and germination of encapsulated and dehydrated carrot somatic embryos

In order to obtain dry artificial seeds, carrot somatic embryos were pre-treated before being encapsulated into calcium-alginate-gellan gum, and slowly dehydrated unitil 15% RH (relative humidity of the chamber). ABA (1 to 10 μM), 1 to 5 mM proline, an osmotic pressure of ±520 mOsm, or heat (35°C) enhanced the desiccation tolerance of encapsulated somatic embryos. Some treatments were complementary, like 10 μM ABA and 10% sucrose, 10 μM ABA and heat (35°C), or 10% sucrose and cold (4°C). In such conditions, complete or almost total (95.6–100% germination) desiccation tolerance was then obtained. These treatments may act by the acquisition of sufficient-and well-balanced-protein and starch reserves. osmotic treatments, ABA, and above all proline, promoted protein accumulation, meanwhile starch reserves were slightly depleted by 10–20 μM ABA, proline, and poor sucrose-osmotic treatments (8% trehalose). All the treatments were found to enhance viability during dehydration, as observed by fluorescence. Sucrose may be partly replaced by other osmotica. Alone, it has a negative effect on the depletion of starch reserves. Cold (4°C) with 10% sucrose may favor the glassy state transition. ABA and proline appear to be involved in the same process leading to the acquisition of partial desiccation tolerance. Heat (35°C), or 10% sucrose, have been found to complement ABA action in the acquisition of full desiccation tolerance.     

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.     

Inhibitory cholinergic control of endogenous GABA release from electrically stimulated cortical slices and K-depolarized synaptosomes

In the present study we characterize the optimal experimental conditions under which to investigate the cholinergic regulation of endogenous electrically evoked γ-aminobutyric acid (GABA) release from guinea pig cortical slices. Superfusion with the neuronal GABA reuptake inhibitor, SKF89976A (10 μM) caused cortical GABA release to be linearly correlated with the frequency of electrical stimulation (5, 10, 20 Hz). Electrically evoked GABA release (10 Hz) was tetrodotoxin-sensitive and Ca²⁺-dependent and was under GABA_B autoreceptor control. Under these experimental conditions, acetylcholine (0.1–10 μM) and physostigmine (30 μM) decreased the electrically evoked GABA release while the M₂ receptor antagonist AFDX-116 (0.01–0.1 μM) counteracted these effects. Similar results were also observed in a cortical synaptosomal preparation stimulated with K⁺ (10 mM). These findings demonstrate an inhibitory cholinergic regulation of electrically evoked GABA release via M₂ receptors located on cortical GABAergic terminals.     

Effects of CRF, AVP and opioid peptides on pituitary-adrenal responses in sheep

Intravenous administration of equimolar doses of CRF (30 μg) and AVP (6 μg) to mature female sheep resulted in elevated plasma concentrations of ACTH and cortisol. Simultaneous administration of equimolar amounts of CRF and AVP resulted in a greater ACTH response compared with the sum of the responses to CRF or AVP given independently. Intravenous bolus administration of the endogenous opioid, Met-enkephalin (2.5 mg), and its potent and long-acting analogue, [D-Ala²,N-Phe⁴,Met(O)ol⁵]-enkephalin [FK33–824 (250 μg)], did not alter ACTH or cortisol secretion. Furthermore, naloxone, an opioid receptor antagonist given alone or concurrently with Met-enkephalin or FK33–824, was without effect. Pituitary-adrenal responses to CRF were unaltered by simultaneous administration of Met-enkephalin, FK33–824 or naloxone. These results suggest that in the sheep, opioid involvement in the tonic regulation of pituitary-adrenal function is absent. However, CRF and AVP may act alone or in synergy to control the release of biologically active ACTH from the sheep pituitary gland.     

Decreased dynorphin levels and increased kappa opioid receptor binding in male rats with isolation-induced hypertension

The role of hippocampal dynorphin A (1–8) (Dyn A (1–8)) and kappa opioid receptors was investigated in the isolation-induced hypertensive rat (IHR). Male Sprague Dawley rats were either isolated (1 per cage) or grouped (3 per cage) for 7 days. Isolated rats exhibited increased blood pressure (systolic blood pressure 159.7 ± 6.6 mmHg) whereas the grouped rats remained normotensive (systolic blood pressure 137 ± 6.3 mmHg). Using radioligand binding techniques we observed a significant increase in kappa opioid receptor binding in the hippocampus of isolated rats (56% increase) and this further increased when the length of isolation was increased to 2 weeks (72% increase). Radioimmunoassay showed that isolation decreased the hippocampal content of Dyn A (1–8) from 12.7 ± 0.4 to 11.6 ± 0.2 pg Dyn A (1–8) per 10 mg tissue (rats weighing approximately 100 g) and from 13.3 ± 0.8 to 9.7 ± 1 pg Dyn A (1–8) per 10 mg tissue (approximately 200 g rats). These data suggest that functional alterations in the hippocampal dynorphin system may be involved in the maintenance of isolation induced hypertension.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.