tetrodotoxin and resiniferatoxin

Topics: Animals; Diterpenes; Drug Implants; Female; Hyperalgesia; Male; Neuralgia; Neuralgia, Postherpetic; Neurotoxins; Rats; Rats, Sprague-Dawley; Tetrodotoxin

The present study investigated the influence of intravesically instilled resiniferatoxin (RTX) or tetrodotoxin (TTX) on the distribution, number, and chemical coding of noradrenergic and cholinergic nerve fibers (NF) supplying the urinary bladder in female pigs. Samples from the bladder wall were processed for double-labelling immunofluorescence with antibodies against cholinergic and noradrenergic markers and some other neurotransmitter substances. Both RTX and TTX caused a significant decrease in the number of cholinergic NF in the urinary bladder wall (in the muscle coat, submucosa, and beneath the urothelium). RTX instillation resulted in a decrease in the number of noradrenergic NF in the submucosa and urothelium, while TTX treatment caused a significant increase in the number of these axons in all the layers. The most remarkable changes in the chemical coding of the NF comprised a distinct decrease in the number of the cholinergic NF immunoreactive to CGRP (calcitonin gene-related peptide), nNOS (neuronal nitric oxide synthase), SOM (somatostatin) or VIP (vasoactive intestinal polypeptide), and an increase in the number of noradrenergic NF immunopositive to GAL (galanin) or nNOS, both after RTX or TTX instillation. The present study is the first to suggest that both RTX and TTX can modify the number of noradrenergic and cholinergic NF supplying the porcine urinary bladder.

Topics: Adrenergic Fibers; Animals; Calcitonin Gene-Related Peptide; Cholinergic Fibers; Diterpenes; Female; Galanin; Neuropeptide Y; Nitric Oxide Synthase Type I; Somatostatin; Swine; Tetrodotoxin; Urinary Bladder; Vasoactive Intestinal Peptide; Vesicular Acetylcholine Transport Proteins

Vanilloids, high temperature, and low pH activate the transient receptor potential vanilloid type 1 (TRPV1) receptor. In spinal dorsal root ganglia, co-activation of one of these gating sites on TRPV1 sensitized receptor gating by other modes. Here in rat brainstem slices, we examined glutamate synaptic transmission in nucleus of the solitary tract (NTS) neurons where most cranial primary afferents express TRPV1, but TRPV1 sensitization is unknown. Electrical shocks to the solitary tract (ST) evoked EPSCs (ST-EPSCs). Activation of TRPV1 with capsaicin (100 nM) increased spontaneous EPSCs (sEPSCs) but inhibited ST-EPSCs. High concentrations of the ultra-potent vanilloid resiniferatoxin (RTX, 1 nM) similarly increased sEPSC rates but blocked ST-EPSCs. Lowering the RTX concentration to 150 pM modestly increased the frequency of the sEPSCs without causing failures in the evoked ST-EPSCs. The sEPSC rate increased with raising bath temperature to 36 °C. Such thermal responses were larger in 150 pM RTX, while the ST-EPSCs remained unaffected. Vanilloid sensitization of thermal responses persisted in TTX but was blocked by the TRPV1 antagonist capsazepine. Our results demonstrate that multimodal activation of TRPV1 facilitates sEPSC responses in more than the arithmetic sum of the two activators, i.e. co-activation sensitizes TRPV1 control of spontaneous glutamate release. Since action potential evoked glutamate release is unaltered, the work provides evidence for cooperativity in gating TRPV1 plus a remarkable separation of calcium mechanisms governing the independent vesicle pools responsible for spontaneous and evoked release at primary afferents in the NTS.

Topics: Animals; Capsaicin; Diterpenes; Electric Stimulation; Excitatory Postsynaptic Potentials; Glutamic Acid; In Vitro Techniques; Male; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sensory System Agents; Sodium Channel Blockers; Solitary Nucleus; Synaptic Transmission; Tetrodotoxin; TRPV Cation Channels

Both resiniferatoxin (RTX) and tetrodotoxin (TTX) have been reported to be effective in several urinary bladder dysfunction clinical trials. The aim of this study was to establish the effect of intravesical administration of RTX and TTX on neuropeptides Y (NPY) and tyrosine hydroxylase (TH) relationship in the paracervical ganglion (PCG) neurons supplying the urinary bladder in the pig. TH is an enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to dihydroxyphenylalanine (DOPA) and is used as a marker of catecholaminergic neurons. NPY augments the vasoconstrictor effects of noradrenergic neurons, and is involved in pathophysiological processes as a neuromodulator. To identify the PCG neurons supplying urinary bladder Fast Blue (FB) was injected into the bladder wall prior to intravesical RTX or TTX administration. Consequent application of immunocytochemical methods revealed that in control group 64.08 % of FB-positive PCG neurons contain NPY and 4.25 % TH. Intravesical infusion of RTX resulted upregulation of the NPY-IR neurons to 82.97 % and TH-IR to 43.78 %. Also administration of TTX induced further increase number of TH-IR neurons to 77.49 % but induced decrease number of NPY-IR neurons to 57.45 %. Both neurotoxins affect chemical coding of the PCG neural somata supplying urinary bladder, but the effects of their action are different. This results shed light on possible involvement of RTX and TTX on curing tissue, and potentially could help us to broaden our neurourological armamentarium.

Topics: Animals; Diterpenes; Female; Ganglia, Sympathetic; Neurons; Neuropeptide Y; Neurotoxins; Swine; Tetrodotoxin; Tyrosine 3-Monooxygenase; Urinary Bladder

The aim of the present study was to establish the effect of intravesical administration of resiniferatoxin (RTX) and tetrodotoxin (TTX) on the chemical coding of paracervical ganglion (PCG) neurons supplying the urinary bladder in the pig. In order to identify the PCG neurons innervating the bladder, retrograde tracer Fast Blue was injected into the bladder wall prior to intravesical RTX or TTX administration. Consequent application of immunocytochemical methods revealed that in the control group 76.82% of Fast Blue positive PCG neurons contain nitric oxide synthetase (nNOS), and 66.92% contain acetylcholine transferase (ChAT). Intravesical infusion of RTX resulted in a reduction of the nNOS-IR neurons to 57.74% and ChAT-IR to 57.05%. Alternative administration of TTX induced an increase of nNOS-IR neurons up to 79.29% and a reduction of the ChAT-IR population down to 3.73% of the Fast Blue positive PCG cells. Our data show that both neurotoxins affect the chemical coding of PCG cells supplying the porcine urinary bladder, but the effects of their action are different. Moreover, these results shed light on the possible involvement of NO-ergic and cholinergic neurons in the mechanisms of therapeutic action exerted by RTX and TTX in curing the overactive bladder disorder.

Topics: Animals; Choline O-Acetyltransferase; Diterpenes; Female; Ganglia, Autonomic; Neurons; Nitric Oxide Synthase Type I; Protein Transport; Swine; Tetrodotoxin; Urinary Bladder

Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs), co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG) neurons expressing the transient receptor potential vanilloid-1 (TRPV1) receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX) prior to inflammation and behavioural testing.. CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs.. Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned animals. Therefore, these data suggest that CANs contribute to visceral hypersensitivity during inflammation.

Topics: Animals; Behavior, Animal; Colon; Diterpenes; Ganglia, Spinal; Hypersensitivity; Inflammation; NAV1.9 Voltage-Gated Sodium Channel; Neurons; Neurons, Afferent; Neuropeptides; Organ Specificity; Protein Subunits; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Receptors, N-Methyl-D-Aspartate; Sodium Channels; Tetrodotoxin; Trinitrobenzenesulfonic Acid; TRPV Cation Channels; Viscera

In the present study we characterized the receptor(s) that mediates non-adrenergic non-cholinergic (NANC) contractions of isolated guinea pig cervical trachea, using CP-99,994, a selective neurokinin (NK1) receptor antagonist, and SR-48,968, a selective neurokinin (NK2) receptor antagonist. The activity of these two antagonists was determined against contractions to the selective agonists ([beta Ala8]NKA(4-10) for NK2 and [Sar9,Met(O2)11]SP for NK1) and the nonselective (SP and NKA) NK receptor agonists. CP-99,994 was inactive versus NKA and [beta Ala8]NKA(4-10) but antagonized SP- and [Sar9,Met(O2)11]SP-induced contractions with -log KB values of 5.6 +/- 0.2 and 7.7 +/- 0.2, respectively. SR-48,968 was inactive versus SP and [Sar9,Met(O2)11]SP but was active versus NKA and [beta Ala8]NKA(4-10), yielding -log KB values of 8.4 +/- 0.2 and 9.1 +/- 0.2, respectively. In the presence of 1 microM atropine, 1.4 microM indomethacin, 0.2 microM timolol, and 4 microM thiorphan, electrical field stimulation (16 Hz, 2.0 ms, 50 V for 10 every 30 min) elicited a NANC contractile response which was not significantly altered by CP-99,994 (3 microM) or the nitric oxide synthase inhibitor L-NAME (10 microM) but was completely inhibited by tetrodotoxin (TTX) (1 microM) and was also reduced to 58 +/- 12, 31 +/- 16, 8 +/- 4, and 0% of control by 15, 50, 150, and 1500 nM SR-48,968, respectively. Resiniferatoxin (1 and 10 nM) produced a well-maintained concentration-dependent contraction, which was 57.8 +/- 4.8 and 61.6 +/- 3.8%, respectively, of the carbachol-induced maximum response. Contractions were not significantly modified by L-NAME and were not blocked by TTX (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Airway Resistance; Animals; Arginine; Benzamides; Diterpenes; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Male; Neurotoxins; NG-Nitroarginine Methyl Ester; Piperidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Tetrodotoxin; Trachea

Whole cell currents evoked by pain-inducing agents--bradykinin (Bk), capsaicin (Cap), and reciniferatoxin (RTX), and their modulation of voltage-activated Ca currents were examined in F-11 cells using a patch electrode voltage clamp technique. Most F-11 cells generated action potentials under current clamp if their membrane potentials were held sufficiently negative. Average peak inward Na current (INa) was 100 microA/cm2 and the INa was abolished by 10(-6) M tetrodotoxin. At least two types of Ca currents could be clearly distinguished on the basis of voltage dependency and kinetics; a low threshold transient ICa(t) and a high threshold sustained ICa(l). In addition, another high threshold transient Ca current, presumably ICa(n), was observed. About 30% of the cells produced inward current for these pain-inducing agents, when activated at the membrane holding potential of -70 mV. In some F-11 cells, the amplitude of action potential was observed to increase during 10(-6) M Cap-induced depolarization. Both low and high threshold Ca currents were reduced by 10(-6) M Bk in the majority of the cells. Similarly, both 10(-6) M Cap and 10(-9) M RTX reduced these Ca currents. However, a considerable number of cells showed an initial enhancement followed by reduction in the amplitude of these Ca currents. With higher concentrations of these ligands, all Ca currents were suppressed. Such modulation of voltage-activated Ca currents by pain-inducing agents occurred in both the presence and absence of apparent receptor-activated current flows in the cells. In pertussis toxin (PTX)-treated cells, the inhibitory modulation of Ca currents by pain-inducing agents was suppressed. In contrast, in cholera toxin (CTX)-treated cells, this inhibitory modulation appeared to be enhanced. These data indicate that the inhibitory modulation of Ca channel currents by Cap and RTX, similarly to that of Bk, involves a PTX-sensitive inhibitory G protein (Gi).

Topics: Action Potentials; Animals; Bradykinin; Calcium Channel Blockers; Calcium Channels; Capsaicin; Cell Line; Diterpenes; Electrophysiology; Ganglia, Spinal; Mice; Neurons; omega-Conotoxins; Pain; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Receptors, Bradykinin; Receptors, Neurotransmitter; Tetrodotoxin

To study the role of the axon reflex in resiniferatoxin (RTX)-induced bronchoconstriction in vivo, 32 guinea pigs weighing 292 +/- 7 g were randomly divided into five groups: Group 1, control (n = 6); Group 2, chlorisondamine (n = 6); Group 3, tetrodotoxin (TTX, n = 6); Group 4, local capsaicin application (n = 6); and Group 5, systemic capsaicin application (n = 8). Chlorisondamine was used to interrupt ganglionic transmission while TTX was employed to block nerve impulse conduction. In Group 4, capsaicin was locally applied to both cervical vagus nerves 30 min prior to the study whereas capsaicin was given subcutaneously for 5 days starting 9 days before the study in Group 5. Each animal was anesthesized with pentobarbital sodium, cannulated with a tracheal cannula and venous catheter, paralyzed with gallamine triethiodide, and artificially ventilated. All the above animals were treated with atropine (0.2 mg/kg) and phenoxybenzamine (0.5 mg/kg). Resiniferatoxin (2 micrograms/kg) was injected intravenously to induce airway constriction. Immediately upon injection of RTX (at 1 min), each animal in the control group exhibited decreases in maximal expiratory flow, dynamic respiratory compliance, and total lung capacity, indicating severe bronchoconstriction. Then the airway spasm ameliorated gradually with time. Animals in Groups 3 and 4 indicated partial abolishment, while those in Group 5 showed complete abolishment, of the RTX-induced bronchoconstriction. On the other hand, the animals in Group 2 did not display any significant alteration in the RTX-induced bronchospasm. Furthermore, we tested RTX-induced bronchoconstriction in 5 additional animals not pretreated with either atropine or phenoxybenzamine. Compared with the data above, no significant differences in RTX-induced respiratory changes were found. Since it is known that TTX blocks nerve conduction, the data suggest that the TTX-sensitive reflex (the axon reflex) via afferent C-fibers plays a significant role in the RTX-induced bronchoconstriction, which is apparently mediated via tachykinins.

Topics: Analysis of Variance; Animals; Axons; Bronchoconstriction; Capsaicin; Chlorisondamine; Diterpenes; Dose-Response Relationship, Drug; Forced Expiratory Volume; Guinea Pigs; Lung Compliance; Male; Maximal Expiratory Flow Rate; Neural Conduction; Reflex; Tetrodotoxin; Time Factors; Total Lung Capacity; Vagus Nerve; Vital Capacity

Vagal nerve stimulation (1 Hz for 1 min), capsaicin (10(-8) M and 10(-6) M), resiniferatoxin (3 x 10(-10) M) and nicotine (10(-4) M) evoked a non-cholinergic bronchoconstriction in the isolated perfused guinea-pig lung preparation. Simultaneously there was an increase in the perfusate levels of calcitonin gene-related peptide-like immunoreactivity, suggesting release from sensory nerves. Both the bronchoconstriction and peptide release evoked by a low concentration of capsaicin (10(-8) M) and that evoked by nerve stimulation were depressed by tetrodotoxin, suggesting involvement of Na+ channel dependent depolarization. Since the effects of capsaicin (10(-8) M) and vagal nerve stimulation were inhibited by omega-conotoxin but not influenced by nifedipine, the Ca(2+)-channel dependent is probably of N-type. Furthermore, the capsaicin analogue resiniferatoxin also evoked omega-conotoxin sensitive peptide release and bronchoconstriction. At the higher capsaicin concentration (10(-6) M), the functional response was only slightly inhibited by omega-conotoxin or tetrodotoxin indicating that capsaicin at this concentration evoked peptide release and functional effects through other mechanisms, probably involving Ca2+ fluxes in the non-selective cation channel associated with the proposed capsaicin receptor. The nicotine (10(-4) M) evoked peptide release and bronchoconstriction were only marginally influenced by omega-conotoxin or tetrodotoxin. It is concluded that the ion-channel mechanisms underlying the peptide releasing properties of antidromic nerve stimulation and low concentrations of capsaicin are similar and depend on action potential propagation, whereas capsaicin in high, toxic concentration and nicotine mainly act via receptor operated channels.

Topics: Animals; Calcitonin Gene-Related Peptide; Calcium Channel Blockers; Capsaicin; Diterpenes; Electric Stimulation; Female; Guinea Pigs; In Vitro Techniques; Ion Channels; Lung; Male; Nerve Endings; Neurokinin A; Neurons, Afferent; Nicotine; omega-Conotoxins; Peptides; Peptides, Cyclic; Tetrodotoxin; Vagus Nerve