Big conductance calcium-activated potassium channel openers control spasticity without sedation.

BACKGROUND AND PURPOSE: Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity. EXPERIMENTAL APPROACH: Following the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans. KEY RESULTS: VSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity. CONCLUSIONS AND IMPLICATIONS: We identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity.

Effects of in vitro, acute and chronic treatment with fluoxetine on the sympathetic neurotransmission of rat vas deferens.

It is described that fluoxetine treatment is able to induce ejaculatory disorders. However, the exact mechanism is still not fully understood. Therefore, this study was carried out to further evaluate the anti-ejaculatory effects of fluoxetine, using different approaches (in vitro or in vivo treatments), on the sympathetic neurotransmission of the rat vas deferens. Vas deferens from male Wistar rats were used to check the in vitro effects of fluoxetine 10-6M, 3.10-6M or 10-5M. Animals were also acutely (20mg/kg, i.p. 4h or 24h) or chronically (10mg/kg, i.p., 30days) treated with fluoxetine or drug-free vehicle. The vas deferens from non-treated and treated animals were isolated and mounted in an isolated organ bath for the study of the contractions induced by adrenergic agonists, tyramine, 5-HT, Ca2+ or electrical field stimulation. In vitro or acute treatment with fluoxetine decreased the contraction induced by agonists, Ca2+ or electrical field stimulation. The chronic treatment with fluoxetine decreased the contractions induced agonists, tyramine or Ca2+, but did not modify the contractions induced by electrical field stimulation. We have shown that in vitro or in vivo fluoxetine treatment is able to alter the sympathetic neurotransmission of the rat vas deferens which could be related to alterations in the calcium signalling.

Effect of Boerhaavia diffusa in experimental prostatic hyperplasia in rats.

OBJECTIVE: Present investigation was undertaken to study the effectiveness of hydroalcoholic extract of roots of Boerhaavia diffusa in experimental benign prostatic hyperplasia (BPH) in rats using various animal models. MATERIALS AND METHODS: BPH in rats was induced by subcutaneous injection of testosterone (5 mg/kg) daily for 28 days. Rats were divided in to five groups (six rats each). A negative control group received arachis oil (1 ml/kg s.c.) and four groups were injected testosterone. These four groups were further divided into reference group (finasteride 1 mg/kg), model group (testosterone), study group A (B. diffusa 100 mg/kg), and study group B (B. diffusa 250 mg/kg). On the 29(th) day, rats were sacrificed and body weight, prostate weight, bladder weight, and serum testosterone level were measured and histological studies were carried out. Further in vitro analysis of B. diffusa extract on contractility of isolated rat vas deferens and prostate gland, produced by exogenously administered agonists were carried out. All results were expressed as mean ± SEM. 0 Data were analyzed by one-way analysis of variance followed by Tukey's test. RESULTS: B. diffusa (100 mg/kg) treatment for 28 days resulted in significant inhibition of prostate growth (P < 0.05). Drug extract did not have significant change on serum testosterone level. Histopathological analysis of prostate gland supported above results. Results of in vitro experiment suggest that extracts had attenuated the contractile responses of isolated vas deferens and prostate gland to exogenously applied agonists. CONCLUSION: The results suggested that treatment with B. diffusa may improve symptoms of disease and inhibit the increased prostate size. In vitro study implies that herbal extracts has the machinery to produce beneficial effect on prostatic smooth muscle, which would relieve the urinary symptoms of disease. B. diffusa could be a potential source of new treatment of prostatic hyperplasia.

An examination of the cardiovascular effects of an 'Irukandji' jellyfish, Alatina nr mordens.

Irukandji syndrome is usually characterized by delayed severe abdominal, back and chest pain associated with autonomic effects including diaphoresis, hypertension and, in severe cases, myocardial injury and pulmonary oedema. It is most often associated with envenoming by the jellyfish Carukia barnesi, but a number of other jellyfish, including Alatina mordens, are now known to produce Irukandji syndrome. In the present study, nematocyst-derived venom from A. nr mordens (150-250 microg/kg, i.v.) produced a long-lasting pressor effect in anaesthetised rats. This pressor response (250 microg/kg, i.v.) was significantly inhibited by prior administration of the alpha-adrenoceptor antagonist prazosin (200 microg/kg, i.v.) but not by CSL box jellyfish antivenom (300 U/kg, i.v.). A. nr mordens venom 250 microg/kg (i.v.) caused marked increases in plasma adrenaline and noradrenaline concentrations following administration in anaesthetised rats. The venom did not contain appreciable amounts of either adrenaline or noradrenaline. A. nr mordens venom (25 microg/ml) produced a contractile response in rat electrically stimulated vas deferens which was markedly reduced in tissues pre-treated with reserpine (0.1mM) or guanethidine (0.1mM). Sodium dodecyl sulphate (SDS)-PAGE analysis showed that A. nr mordens venom is comprised of multiple protein bands ranging from 10 to 200 kDa. Western blot analysis using CSL box jellyfish antivenom indicated several antigenic proteins in A. nr mordens venom, however, it did not detect all proteins present in the venom. This study characterizes the in vitro and in vivo effects of A. nr mordens venom and indicates that the cardiovascular effects are at least partially mediated by endogenous catecholamine release.

Involvement of mu-opioid receptors in antinociception and inhibition of gastrointestinal transit induced by 7-hydroxymitragynine, isolated from Thai herbal medicine Mitragyna speciosa.

7-hydroxymitragynine, a constituent of the Thai herbal medicine Mitragyna speciosa, has been found to have a potent opioid antinociceptive effect. In the present study, we investigated the mechanism of antinociception and the inhibitory effect on gastrointestinal transit of 7-hydroxymitragynine, and compared its effects with those of morphine. When administered subcutaneously to mice, 7-hydroxymitragynine produced antinociceptive effects about 5.7 and 4.4 times more potent than those of morphine in the tail-flick (ED50=0.80 mg/kg) and hot-plate (ED50=0.93 mg/kg) tests, respectively. The antinociceptive effect of 7-hydroxymitragynine was significantly blocked by the mu1/mu2-opioid receptor antagonist beta-funaltrexamine hydrochloride (beta-FNA) and the mu1-opioid receptor-selective antagonist naloxonazine in both tests. Thus, 7-hydroxymitragynine acts predominantly on mu-opioid receptors, especially on mu1-opioid receptors. Isolated tissue studies further supported its specificity for the mu-opioid receptors. Further, 7-hydroxymintragynine dose-dependently (ED50=1.19 mg/kg, s.c.) and significantly inhibited gastrointestinal transit in mice, as morphine does. The inhibitory effect was significantly antagonized by beta-FNA pretreatment, but slightly antagonized by naloxonazine. The ED50 value of 7-hydroxymitragynine on gastrointestinal transit was larger than its antinociceptive ED50 value. On the other hand, morphine significantly inhibits gastrointestinal transit at a much smaller dose than its antinociceptive dose. These results suggest that mu-opioid receptor mechanisms mediate the antinociceptive effect and inhibition of gastrointestinal transit. This compound induced more potent antinociceptive effects and was less constipating than morphine.

Saw palmetto is an indirectly acting sympathomimetic in the rat-isolated prostate gland.

BACKGROUND: To investigate whether saw palmetto that inhibits alpha1-adrenoceptor binding in vitro affects contractility of the rat prostate gland. METHODS: The effects of a commercially available saw palmetto extract were examined on the contractility of rat-isolated prostate glands. The extract was tested in the presence and absence of phentolamine, prazosin, yohimbine, propranolol, hexamethonium, cocaine, desipramine, nifedipine, guanethidine, atropine, and alpha,beta-methylene ATP to evaluate the mechanism of action. Isolated preparations of rat vas deferens and bladder were used for comparison. RESULTS: Unexpectedly, saw palmetto extract caused contractions of the rat prostate gland that could be attenuated by prazosin, phentolamine, nifedipine, guanethidine, cocaine, and desipramine but not by any of the other pharmacological tools. Similar contractile effects were observed in rat-isolated vas deferens preparations but not in rat-isolated bladder preparations. CONCLUSIONS: In the rat prostate gland, saw palmetto extract causes indirect alpha1-adrenoceptor-mediated contractions via the release of noradrenaline from sympathetic neurons.

Structure-activity studies on high affinity NOP-active hexapeptides.

Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G-protein coupled receptor ORL1 (NOP), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiologic functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited number of high-affinity ligands to NOP, and particularly the lack of availability of useful specific antagonists. Herein, we describe the pharmacologic activity of a series of modified amino acid containing modifications of the hexapeptide Ac-RYYRWR-NH2, with high affinity for NOP. These compounds were tested for binding affinity using [3H]N/OFQ binding to human NOP in CHO cells, and functional activity by measuring stimulation of [35S]GTPgammaS-binding in CHO cell membranes. These studies suggest that each Arg of the hexapeptide is required to maintain high-binding affinity. The peptide maintains high affinity if the Tyr2 or Tyr3 are modified, but at least one of these residues must maintain its hydroxyl group or there is a large decrease in intrinsic activity of the peptide.

Nantenine blocks muscle contraction and Ca2+ transient induced by noradrenaline and K+ in rat vas deferens.

The effect of nantenine, an aporphine alkaloid isolated from Ocotea macrophylla H.B.K., was studied on contractions and Ca(2+) translocation induced by noradrenaline, Ca(2+), or K(+) in the isolated rat vas deferens from reserpinized animals. Concentration-response curves of calcium chloride (CaCl(2)) were performed in the vas deferens, in a Ca(2+)-free nutrient solution, using potassium chloride (KCl, 80 mM) as a depolarizing agent. In these conditions, nantenine (2.35 x 10(-4) and 4.7 x 10(-4) M) significantly reduced the maximum contractions (E(max)) of Ca(2+) (IC(50)=2.6 x 10(-4) M) and noradrenaline (IC(50)=2.9 x 10(-4) M). The contractile responses were totally recovered after the withdrawal of nantenine. In addition, experiments performed to measure simultaneously the contraction and the increase of intracellular Ca(2+) induced by noradrenaline (10(-5) M) or KCl (80 mM) showed that nantenine (2.35 x 10(-4) and 4.7 x 10(-4) M) significantly decreased both effects. The results suggest that a reversible block of Ca(2+) entry could be involved on the non-competitive-like antagonism of nantenine in rat vas deferens.

Azadirachta indica adversely affects sperm parameters and fructose levels in vas deferens fluid of albino rats.

Azadirachta indica treatment for 24 days in albino rats resulted in a decrease in the total sperm count, sperm motility, and forward velocity in vas deferens fluid. The percentage of abnormal sperm increased and the fructose content decreased. As diminished levels of fructose parallel androgen deficiency, we conclude that reduced androgen levels resulting from the anti-androgenic property of A. indica leaves probably influences the physiological maturation of sperm.

Globularia alypum L. extracts reduced histamine and serotonin contraction in vitro.

The present study analyses in in vitro models the pharmacological activity of methanol and dichloromethane extracts (1, 10 and 100 microg/mL) obtained from the leaves and stems of Globularia alypum L. Preincubation of the guinea-pig ileum and rat uterus with both extracts produced a dose dependent abolition of the contractile effects of histamine and serotonin, respectively. At the same doses, neither methanol nor dichloromethane extract reduced the contractile effects of acetylcholine on rat duodenum or noradrenaline on rat vas deferens.

Up-regulation of Ca(2+) channels in vas deferens after chronic treatment of newborn rats with nifedipine.

Radioligand binding and contraction techniques were used to verify if L-type Ca(2+) channels are modified in rat vas deferens after treatment with the blocker nifedipine (15 microg), injected at 7, 14, 21 and 28 days after birth. Vas deferens tissue was used 10, 30 and 90 days after the last injection, to verify if modifications are persistent. Binding studies with cell membranes, using [(3)H]isradipine, showed an increase of the density (B(max)) of Ca(2+) channels by more than 60%, after 10 and 30 days, without changes of affinity (K(d)). Maximal contractions (E(max)) of KCl, were increased by 106% and 37%, respectively, after 10 and 30 days, without changes of apparent affinity (pD(2)). After 90 days, the values of B(max), K(d), E(max) and pD(2) were not different from the controls. Differences were also not found for rats injected when adult. It is concluded that treatment of newborn, but not of adult, rats with nifedipine produced a long-lasting, though reversible, up-regulation of L-type Ca(2+) channels.

Structure-activity relationship of [Nphe1]-NC-(1-13)-NH2, a pure and selective nociceptin/orphanin FQ receptor antagonist.

A series of analogs of the ORL1 receptor antagonist [Nphe1]-NC(1-13)-NH2 was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe1]-NC(1-13)-NH2 is high (pKB 6.43) compared with those of all other compounds except [(S)(betaMe)Nphe1]NC(1-13)-NH2 (pK(B) 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pK(B)) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency.

Deltorphin II analogues with 6-hydroxy-2-aminotetralin-2-carboxylic acid in position 1.

Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorphin analogues with altered hydrophobic and stereoelectronic properties. Deltorphin II analogues were synthesized with the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 6-hydroxy-2-aminotetralin-2-carboxylic acid (Hat) instead of Tyr(1) in the message domain. In the radioreceptor-binding studies, in which type-specific tritiated opioid ligands were used, (R)- and (S)-Hat-deltorphins exhibited similar K(i) values, revealing high delta selectivity. The peptides displayed agonist properties in the in vitro bioassay, with IC(50) values in the subnanomolar range in the mouse vas deferens assay and in the micromolar or higher range in the guinea pig ileum assay, again demonstrating a high selectivity toward delta receptors. The agonist property of the new ligands was confirmed by means of [(35)S]GTPgammaS-binding experiments in membranes of the rat frontal cortex.

Opioid receptor agonistic characteristics of mitragynine pseudoindoxyl in comparison with mitragynine derived from Thai medicinal plant Mitragyna speciosa.

We have previously elucidated the opiate-like action of mitragynine, an active principle isolated from the Thai medicinal plant Mitragyna speciosa. In the present study, effects of the related compound, mitragynine pseudoindoxyl on electrically stimulated contraction in guinea pig ileum and mouse vas deferens, and on its binding affinity in the guinea pig brain membranes were studied. Mitragynine pseudoindoxyl inhibited the electrically stimulated ileum and mouse vas deferens contractions in a concentration-dependent manner. In the ileum, the effective concentration is in an nM order, being nearly equivalent to reported concentrations of the micro-opioid receptor agonist [D-Ala2, Met-Phe4, Gly-ol5] enkephalin (DAMGO), and is 100- and 20-fold smaller than those of mitragynine and morphine, respectively. In the vas deferens, it is 35-fold smaller than that of morphine. The inhibitory action of mitragynine pseudoindoxyl in the ileum was antagonized by the non-selective opioid receptor antagonist naloxone and the micro-receptor antagonist naloxonazine. It was also antagonized by the delta-receptor antagonist naltrindole in the vas deferens. Mitragynine pseudoindoxyl showed a similar binding affinity to DAMGO and naltrindole at micro- and delta-receptors, respectively. However, the affinity at kappa-receptors was negligible. The present study demonstrates that mitragynine pseudoindoxyl, a novel alkaloid structurally different from other opioid agonists, acts on opioid receptors, leading to a potent inhibition of electrically stimulated contraction in the ileum through the micro-receptors and in mouse vas deferens through delta-receptors.

Effect of ambrein on smooth muscle responses to various agonists.

The pharmacological effects of ambrein (isolated from ambergris) on the contractile responses induced by some agonists in smooth muscle preparations were investigated. Ambrein in the concentration range of 10, 50 and 250 microg/ml decreased the spontaneous contraction of the isolated rabbit jejunum, rat uterus and guinea-pig vas deferens. Ambrein-induced antagonism to acetylcholine (Ach) in the guinea-pig ileum was abolished when the concentration of calcium chloride in the Tyrode's solution was increased to 5 mM/l. Furthermore, ambrein did not antagonise nicotine-induced contractions in the isolated rabbit jejunum or serotonin-induced contractions in the isolated guinea-pig ileum and vas deferens or the rat uterus. However, ambrein in the concentration range of 10, 50 and 250 microg/ml antagonised prostaglandins (PGs) E2, D2, F2alpha, and oxytocin-induced contractions in the rat uterus in vitro. Ambrein also antagonised (+/-) noradrenaline and (-) adrenaline-induced contractions in the isolated guinea-pig vas deferens. It is concluded that ambrein-induced non-selective dose-dependent antagonism to the effects of some agonists (Ach, adrenaline, noradrenaline, PGs and oxytocin) in some smooth muscles may be due to the ability of this compound to interfere with the mobilisation of extracellular Ca2+ required for muscular contractions induced by these agonists.

P2X receptors bring new structure to ligand-gated ion channels.

P2X receptors are cation-selective ion channels that open on binding to extracellular ATP; they play a role in fast synaptic transmission between neurones, and from autonomic nerves to smooth muscles. Isolation of cDNAs that encode P2X receptors in the smooth muscle of vas deferens and in phaeochromocytoma cells indicates that the receptors are not related to other ligand-gated ion channels. Their overall structure resembles more closely that of epithelial Na+ channels and the proteins that are thought to form mechanosensitive channels in Caenorhabditis elegans. The type of P2X RNA that is found in vas deferens is expressed preferentially by apoptotic thymocytes, and the type of P2X RNA that is found in PC12 cells is abundant in the pituitary gland, suggesting hitherto unsuspected roles for ATP-gated channels in endocrine and immune function.

Transurethral microwave thermal therapy: pathologic findings in the canine prostate.

Microwave irradiation administered by transurethral transducer to the prostate permits focused hyperthermia with resultant tissue ablation; a coding system within the catheter allows urethral preservation. We evaluated the effect of microwave hyperthermia in 13 dogs receiving 48-79 min of focused irradiation (16-45 watts, intraprostatic temperature > 45 degrees C) delivered by a specially-designed transducer with an operator-controlled directional antenna (T3, Urologix Inc., Minneapolis, MN); one other dog had transducer placement without irradiation (sham control). After treatment, the dogs were in good health, voiding well without complications, and were sacrificed after 5-38 days. The prostate and vasa deferentia were removed, fixed in 10% formalin, grossly inspected, cut at 5 mm intervals, and serially sectioned with whole mount sections; representative sections of the adjacent rectum and distal bladder were also obtained. All cases were histologically evaluated with prostatic mapping without knowledge of treatment or time of sacrifice. In the acute phase (5-13 days), the prostate showed sharply circumscribed periurethral coagulative necrosis with hemorrhage; necrosis was also seen in the mucosa and bladder wall of those with transducers placed at the bladder neck. In the subacute phase (17 days), the hemorrhagic necrosis was resolving, often with cystically dilated urethra due to sloughed necrotic tissue; the urothelium was intact. By 24-38 days, the necrosis was in the late stages of resolution, with residual patchy acute and chronic inflammation at the periphery, and frequent persistence of megalourethra. In all cases, the prostatic capsule was intact, the urethral mucosa was preserved, and the vasa deferentia and rectum were normal except for two cases with mild fat necrosis. Microwave irradiation allows precisely localized thermal ablation of prostatic tissue and enlargement of the urethral bore without clinical complications in dogs, offering promise as a therapeutic alternative to surgery in select patients with symptomatic prostatic nodular hyperplasia.

Pre-clinical and clinical pharmacology of selective muscarinic M3 receptor antagonists.

Muscarinic M3 receptor antagonists have therapeutic potential for the treatment of disorders associated with altered smooth muscle contractility or tone. These include irritable bowel syndrome (IBS), chronic obstructive airways disease (COAD) and urinary incontinence. Zamifenacin is a potent muscarinic receptor antagonist on the guinea pig ileum (pA2 value 9.27) with selectivity over M2 receptors in the atria (135-fold) and M1/M4 receptors in the rabbit vas deferens (78-fold). In addition, zamifenacin had lower affinity for the M3 receptor in the salivary gland (pKi 7.97). In animals, zamifenacin potently inhibited gut motility in the absence of cardiovascular effects and with selectivity over inhibition of salivary secretion. In healthy volunteers, zamifenacin inhibited small and large bowel motility and increased the rate of gastric emptying over a dose range which was associated with minimal anticholinergic side effects. These data show that zamifenacin, a selective muscarinic M3 receptor antagonist, was well tolerated in man and was efficacious as an inhibitor of gut motility. Further studies in patients are required with muscarinic M3 receptor antagonists to confirm efficacy against symptoms in diseases associated with altered smooth muscle contractility.

Laparoscopic implantation of electrodes for stimulation of the hypogastric nerve and the vas deferens in dogs.

The minimal invasiveness of laparoscopy prompted us to investigate its use for placement of electrodes for stimulation of the hypogastric nerve and the vas deferens. In an acute canine model, pressure changes in the vas deferens secondary to electrostimulation were recorded. The laparoscopic approach included four ports. Monopolar cuff electrodes were placed around the hypogastric nerve and the vas deferens, and the leads of both were pulled through the abdominal wall. After multiple percutaneous stimulations (20 mAmp., 20 Hz. and 200 microsecond pulse width), the abdomen was opened through a midline incision to check the position of the electrodes. Electrostimulation was repeated with the abdomen open. Electrostimulation of both the hypogastric nerve and vas deferens resulted in marked pressure rises in the vas. These increases were similar in response to both surface and percutaneous stimulation. We thus believe that laparoscopic implantation of electrodes followed by percutaneous preliminary stimulation of the superior hypogastric plexus or the vas deferens may be a viable future approach to anejaculation.