Polymeric Prodrugs using Dynamic Covalent Chemistry for Prolonged Local Anesthesia.

Depot-type drug delivery systems are designed to deliver drugs at an effective rate over an extended period. Minimizing initial "burst" can also be important, especially with drugs causing systemic toxicity. Both goals are challenging with small hydrophilic molecules. The delivery of molecules such as the ultrapotent local anesthetic tetrodotoxin (TTX) exemplifies both challenges. Toxicity can be mitigated by conjugating TTX to polymers with ester bonds, but the slow ester hydrolysis can result in subtherapeutic TTX release. Here, we developed a prodrug strategy, based on dynamic covalent chemistry utilizing a reversible reaction between the diol TTX and phenylboronic acids. These polymeric prodrugs exhibited TTX encapsulation efficiencies exceeding 90 % and the resulting polymeric nanoparticles showed a range of TTX release rates. In vivo injection of the TTX polymeric prodrugs at the sciatic nerve reduced TTX systemic toxicity and produced nerve block lasting 9.7±2.0 h, in comparison to 1.6±0.6 h from free TTX. This approach could also be used to co-deliver the diol dexamethasone, which prolonged nerve block to 21.8±5.1 h. This work emphasized the usefulness of dynamic covalent chemistry for depot-type drug delivery systems with slow and effective drug release kinetics.

Multi-Omics Analysis of Gene and Protein Candidates Possibly Related to Tetrodotoxin Accumulation in the Skin of Takifugu flavidus.

Pufferfish is increasingly regarded by many as a delicacy. However, the tetrodotoxin (TTX) that accumulates in its body can be lethal upon consumption by humans. TTX is known to mainly accumulate in pufferfish skin, but the accumulation mechanisms are poorly understood. In this study, we aimed to explore the possible mechanism of TTX accumulation in the skin of the pufferfish Takifugu flavidus following treatment with TTX. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we detected 37.3% of toxin accumulated in the skin at the end of the rearing period (168 h). Transcriptome and proteome analyses revealed the mechanism and pathways of TTX accumulation in the skin of T. flavidus in detail. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes analyses strongly suggest that cardiac muscle contraction and adrenergic signaling in cardiomyocyte pathways play an important role in TTX accumulation. Moreover, some upregulated and downregulated genes, which were determined via RNA-Seq, were verified with qPCR analysis. This study is the first to use multi-omics profiling data to identify novel regulatory network mechanisms of TTX accumulation in the skin of pufferfish.

A Microencapsulation Method for Delivering Tetrodotoxin to Bivalves to Investigate Uptake and Accumulation.

Most marine biotoxins are produced by microalgae. The neurotoxin tetrodotoxin (TTX) has been reported in many seafood species worldwide but its source is unknown, making accumulation and depuration studies in shellfish difficult. Tetrodotoxin is a water-soluble toxin and cannot be directly ingested by shellfish. In the present study, a method was developed which involved binding TTX to solid particles of humic acid and encapsulating them in agar-gelatin capsules. A controlled quantity of TTX-containing microcapsules (size range 20-280 µm) was fed to Paphies australis, a bivalve known to accumulate TTX in the wild. The TTX-containing microcapsules were fed to P. australis every second day for 13 days. Ten P. australis (including five controls fed non-toxic microalgae) were harvested after 7 days and ten after 13 days. Paphies australis accumulated TTX, reaching concentrations of up to 103 µg kg-1 by day 13, exceeding the European Food Safety Authority recommended concentration of 44 µg kg-1 in shellfish. This novel method will allow future studies to explore the effects, accumulation and depuration rates of TTX in different animals and document how it is transferred through food webs.

A neural circadian signal essential for ovulation is generated in the suprachiasmatic nucleus during each stage of the oestrous cycle.

NEW FINDINGS: What is the central question of this study? Is the suprachiasmatic nucleus the structure that generates the neural circadian signals that occur during every stage of the oestrous cycle, not only pro-oestrus, and are these signals essential for proper regulation of ovulation? What is the main finding and its importance? Transient inhibition of Na+ -dependent action potentials in the suprachiasmatic nucleus by tetrodotoxin microinjection at 14.00 h inhibits ovulation irrespective of the stage of the oestrous cycle at which the procedure is performed. Microinjection of saline solution into the suprachiasmatic nucleus has a disruptive effect on ovulation that depends on the stage of the cycle at which it is administered. ABSTRACT: Reproduction is a highly timed process that depends on both the reproductive and circadian systems. The core oscillator of the latter resides at the suprachiasmatic nuclei (SCN) and it is pivotal for the regulation of the pro-oestrus pre-ovulatory surge of gonadotropins in females. There is evidence to suggest that this system may be involved in the regulation of neuroendocrine events that are essential for ovulation and that occur prior to pro-oestrus. We explored this possibility by transiently inactivating the SCN. Female rats were implanted with guide cannulas aimed at the SCN. After recovery of the oestrous cycle, animals were injected with tetrodotoxin (TTX), artificial cerebrospinal fluid (ACSF) or saline solution while freely moving. Injections were performed at 14.00 h of each stage of the oestrous cycle. Animals were killed on the next predicted oestrus day, the number of ova shed was counted and intact rats at oestrus stage were used as absolute control. ACSF did not modify ovulation. Saline solution blocked ovulation in oestrus- and dioestrus-injected rats. Irrespectively of the stage of the oestrous cycle, TTX blocked ovulation. These results lead us to suggest that a neural circadian signal, pivotal for triggering the gonadotropin pre-ovulatory surge, arises from the SCN during the critical window of pro-oestrus. We also suggest that a similar signal, needed for the regulation of other events that are indispensable for proper regulation of ovulation, is also generated in this nucleus during the other stages of the cycle at a similar time.

Graded Transmission without Action Potentials Sustains Rhythmic Activity in Some But Not All Modulators That Activate the Same Current.

Neurons in the central pattern-generating circuits in the crustacean stomatogastric ganglion (STG) release neurotransmitter both as a graded function of presynaptic membrane potential that persists in TTX and in response to action potentials. In the STG of the male crab Cancer borealis, the modulators oxotremorine, C. borealis tachykinin-related peptide Ia (CabTRP1a), red pigment concentrating hormone (RPCH), proctolin, TNRNFLRFamide, and crustacean cardioactive peptide (CCAP) produce and sustain robust pyloric rhythms by activating the same modulatory current (IMI), albeit on different subsets of pyloric network targets. The muscarinic agonist oxotremorine, and the peptides CabTRP1a and RPCH elicited rhythmic triphasic intracellular alternating fluctuations of activity in the presence of TTX. Intracellular waveforms of pyloric neurons in oxotremorine and CabTRP1a in TTX were similar to those in the intact rhythm, and phase relationships among neurons were conserved. Although cycle frequency was conserved in oxotremorine and TTX, it was altered in CabTRP1a in the presence of TTX. Both rhythms were primarily driven by the pacemaker kernel consisting of the Anterior Burster and Pyloric Dilator neurons. In contrast, in TTX the circuit remained silent in proctolin, TNRNFLRFamide, and CCAP. These experiments show that graded synaptic transmission in the absence of voltage-gated Na+ current is sufficient to sustain rhythmic motor activity in some, but not other, modulatory conditions, even when each modulator activates the same ionic current. This further demonstrates that similar rhythmic motor patterns can be produced by qualitatively different mechanisms, one that depends on the activity of voltage-gated Na+ channels, and one that can persist in their absence.SIGNIFICANCE STATEMENT The pyloric rhythm of the crab stomatogastric ganglion depends both on spike-mediated and graded synaptic transmission. We activate the pyloric rhythm with a wide variety of different neuromodulators, all of which converge on the same voltage-dependent inward current. Interestingly, when action potentials and spike-mediated transmission are blocked using TTX, we find that the muscarinic agonist oxotremorine and the neuropeptide CabTRP1a sustain rhythmic alternations and appropriate phases of activity in the absence of action potentials. In contrast, TTX blocks rhythmic activity in the presence of other modulators. This demonstrates fundamental differences in the burst-generation mechanisms in different modulators that would not be suspected on the basis of their cellular actions at the level of the targeted current.

Transtympanic Delivery of Local Anesthetics for Pain in Acute Otitis Media.

Acute otitis media (AOM) commonly causes pain and distress in children. Existing analgesic ototopical drops have limited effectiveness due to the impermeable nature of the tympanic membrane. We developed a local drug delivery system to provide sustained pain relief in patients with AOM, achieved by applying a single dose of a hydrogel formulation onto the tympanic membrane. Successful drug delivery across intact tympanic membranes was demonstrated using the amino-amide anesthetic, bupivacaine, and a highly potent site 1 sodium channel blocker anesthetic, tetrodotoxin. The chemical permeation enhancers incorporated in the delivery system increased the permeability of the tympanic membrane to the anesthetics considerably. The drug levels measured using a previously developed ex vivo model reflect the potential for highly effective local anesthesia.

The Duration of Nerve Block from Local Anesthetic Formulations in Male and Female Rats.

PURPOSE: It is unknown whether there are sex differences in response to free or encapsulated local anesthetics. METHODS: We examined nerve block duration and toxicity following peripheral nerve blockade in male and female rats. We studied the local anesthetic bupivacaine (free or encapsulated) as well as tetrodotoxin, which acts on a different site of the same voltage-gated channel. RESULTS: Sensory nerve blockade was 158.5 [139-190] minutes (median [interquartile range]) (males) compared to 173 [134-171] minutes (females) (p = 0.702) following bupivacaine injection, N = 8 male, 8 female. Motor nerve blockade was 157 [141-171] minutes (males) compared to 172 [146-320] minutes (females) (p = 0.2786). Micellar bupivacaine (N = 8 male, 8 female) resulted in sensory nerve blockade of 266 [227-320] minutes (males) compared to 285 [239-344] minutes (females) (p = 0.6427). Motor nerve blockade was 264 [251-264] minutes (males) compared to 287 [262-287] minutes (females) (p = 0.3823). Liposomal bupivacaine (N = 8 male, 8 female) resulted in sensory nerve blockade of 240 [207-277] minutes (males) compared to 289 [204-348] minutes (females) (p = 0.1654). Motor nerve blockade was 266 [237-372] minutes (males) compared to 317 [251-356] minutes (females) (p = 0.6671). Following tetrodotoxin injection (N = 12 male,12 female) sensory nerve blockade was 54.8 [5-117] minutes (males) compared to 54 [14-71] minutes (females) (p = 0.6422). Motor nerve blockade was 72 [40-112] minutes (males) compared to 64 [32-143] minutes (females) (p = 0.971). CONCLUSIONS: We found no statistically significant sex differences associated with the formulations tested. In both sexes, durations of nerve block were similar between micellar and liposomal bupivacaine formulations, despite the micellar formulation containing less drug.

Prolonged Duration Local Anesthesia by Combined Delivery of Capsaicin- and Tetrodotoxin-Loaded Liposomes.

BACKGROUND: Capsaicin, the active component of chili peppers, can produce sensory-selective peripheral nerve blockade. Coadministration of capsaicin and tetrodotoxin, a site-1 sodium channel blocker, can achieve a synergistic effect on duration of nerve blocks. However, capsaicin can be neurotoxic, and tetrodotoxin can cause systemic toxicity. We evaluated whether codelivery of capsaicin and tetrodotoxin liposomes can achieve prolonged local anesthesia without local or systemic toxicity. METHODS: Capsaicin- and tetrodotoxin-loaded liposomes were developed. Male Sprague-Dawley rats were injected at the sciatic nerve with free capsaicin, capsaicin liposomes, free tetrodotoxin, tetrodotoxin liposomes, and blank liposomes, singly or in combination. Sensory and motor nerve blocks were assessed by a modified hotplate test and a weight-bearing test, respectively. Local toxicity was assessed by histologic scoring of tissues at the injection sites and transmission electron microscopic examination of the sciatic nerves. Systemic toxicity was assessed by rates of contralateral nerve deficits and/or mortality. RESULTS: The combination of capsaicin liposomes and tetrodotoxin liposomes achieved a mean duration of sensory block of 18.2 hours (3.8 hours) [mean (SD)], far longer than that from capsaicin liposomes [0.4 hours (0.5 hours)] (P < .001) or tetrodotoxin liposomes [0.4 hours (0.7 hours)] (P < .001) given separately with or without the second drug in free solution. This combination caused minimal myotoxicity and muscle inflammation, and there were no changes in the percentage or diameter of unmyelinated axons. There was no systemic toxicity. CONCLUSIONS: The combination of encapsulated tetrodotoxin and capsaicin achieved marked prolongation of nerve block. This combination did not cause detectable local or systemic toxicity. Capsaicin may be useful for its synergistic effects on other formulations even when used in very small, safe quantities.

Combination Formulation of Tetrodotoxin and Lidocaine as a Potential Therapy for Severe Arrhythmias.

Severe arrhythmias-such as ventricular arrhythmias-can be fatal, but treatment options are limited. The effects of a combined formulation of tetrodotoxin (TTX) and lidocaine (LID) on severe arrhythmias were studied. Patch clamp recording data showed that the combination of LID and TTX had a stronger inhibitory effect on voltage-gated sodium channel 1.5 (Nav1.5) than that of either TTX or LID alone. LID + TTX formulations were prepared with optimal stability containing 1 µg of TTX, 5 mg of LID, 6 mg of mannitol, and 4 mg of dextran-40 and then freeze dried. This formulation significantly delayed the onset and shortened the duration of arrhythmia induced by aconitine in rats. Arrhythmia-originated death was avoided by the combined formulation, with a decrease in the mortality rate from 64% to 0%. The data also suggests that the anti-arrhythmic effect of the combination was greater than that of either TTX or LID alone. This paper offers new approaches to develop effective medications against arrhythmias.

Hollow Silica Nanoparticles Penetrate the Peripheral Nerve and Enhance the Nerve Blockade from Tetrodotoxin.

The efficacy of tetrodotoxin (TTX), a very potent local anesthetic, is limited by its poor penetration through barriers to axonal surfaces. To address this issue, we encapsulated TTX in hollow silica nanoparticles (TTX-HSN) and injected them at the sciatic nerve in rats. TTX-HSN achieved an increased frequency of successful blocks, prolonged the duration of the block, and decreased the toxicity compared to free TTX. In animals injected with fluorescently labeled HSN, the imaging of frozen sections of nerve demonstrated that HSN could penetrate into nerve and that the penetrating ability of silica nanoparticles was highly size-dependent. These results demonstrated that HSN could deliver TTX into the nerve, enhancing efficacy while improving safety.

Sodium Channel Nav1.8 Underlies TTX-Resistant Axonal Action Potential Conduction in Somatosensory C-Fibers of Distal Cutaneous Nerves.

Voltage-gated sodium (NaV) channels are responsible for the initiation and conduction of action potentials within primary afferents. The nine NaV channel isoforms recognized in mammals are often functionally divided into tetrodotoxin (TTX)-sensitive (TTX-s) channels (NaV1.1-NaV1.4, NaV1.6-NaV1.7) that are blocked by nanomolar concentrations and TTX-resistant (TTX-r) channels (NaV1.8 and NaV1.9) inhibited by millimolar concentrations, with NaV1.5 having an intermediate toxin sensitivity. For small-diameter primary afferent neurons, it is unclear to what extent different NaV channel isoforms are distributed along the peripheral and central branches of their bifurcated axons. To determine the relative contribution of TTX-s and TTX-r channels to action potential conduction in different axonal compartments, we investigated the effects of TTX on C-fiber-mediated compound action potentials (C-CAPs) of proximal and distal peripheral nerve segments and dorsal roots from mice and pigtail monkeys (Macaca nemestrina). In the dorsal roots and proximal peripheral nerves of mice and nonhuman primates, TTX reduced the C-CAP amplitude to 16% of the baseline. In contrast, >30% of the C-CAP was resistant to TTX in distal peripheral branches of monkeys and WT and NaV1.9-/- mice. In nerves from NaV1.8-/- mice, TTX-r C-CAPs could not be detected. These data indicate that NaV1.8 is the primary isoform underlying TTX-r conduction in distal axons of somatosensory C-fibers. Furthermore, there is a differential spatial distribution of NaV1.8 within C-fiber axons, being functionally more prominent in the most distal axons and terminal regions. The enrichment of NaV1.8 in distal axons may provide a useful target in the treatment of pain of peripheral origin.SIGNIFICANCE STATEMENT It is unclear whether individual sodium channel isoforms exert differential roles in action potential conduction along the axonal membrane of nociceptive, unmyelinated peripheral nerve fibers, but clarifying the role of sodium channel subtypes in different axonal segments may be useful for the development of novel analgesic strategies. Here, we provide evidence from mice and nonhuman primates that a substantial portion of the C-fiber compound action potential in distal peripheral nerves, but not proximal nerves or dorsal roots, is resistant to tetrodotoxin and that, in mice, this effect is mediated solely by voltage-gated sodium channel 1.8 (NaV1.8). The functional prominence of NaV1.8 within the axonal compartment immediately proximal to its termination may affect strategies targeting pain of peripheral origin.

Repeatable and adjustable on-demand sciatic nerve block with phototriggerable liposomes.

Pain management would be greatly enhanced by a formulation that would provide local anesthesia at the time desired by patients and with the desired intensity and duration. To this end, we have developed near-infrared (NIR) light-triggered liposomes to provide on-demand adjustable local anesthesia. The liposomes contained tetrodotoxin (TTX), which has ultrapotent local anesthetic properties. They were made photo-labile by encapsulation of a NIR-triggerable photosensitizer; irradiation at 730 nm led to peroxidation of liposomal lipids, allowing drug release. In vitro, 5.6% of TTX was released upon NIR irradiation, which could be repeated a second time. The formulations were not cytotoxic in cell culture. In vivo, injection of liposomes containing TTX and the photosensitizer caused an initial nerve block lasting 13.5 ± 3.1 h. Additional periods of nerve block could be induced by irradiation at 730 nm. The timing, intensity, and duration of nerve blockade could be controlled by adjusting the timing, irradiance, and duration of irradiation. Tissue reaction to this formulation and the associated irradiation was benign.

Enhanced Triggering of Local Anesthetic Particles by Photosensitization and Photothermal Effect Using a Common Wavelength.

On-demand pain relief systems would be very helpful additions to the armamentarium of pain management. Near-infrared triggered drug delivery systems have demonstrated the potential to provide such care. However, challenges remain in making such systems as stimulus-sensitive as possible, to enhance depth of tissue penetration, repeatability of triggering, and safety. Here we developed liposomes containing the local anesthetic tetrodotoxin and also containing a photosensitizer and gold nanorods that were excitable at the same near-infrared wavelength. The combination of triggering mechanisms enhanced the photosensitivity and repeatability of the system in vitro when compared with liposomes with a single photoresponsive component. In vivo, on-demand local anesthesia could be induced with a low irradiance and short irradiation duration, and liposomes containing both photosensitizer and gold nanorods were more effective than those containing just one photoresponsive component. Tissue reaction was benign.

Phototriggered Local Anesthesia.

We report a phototriggerable formulation enabling in vivo repeated and on-demand anesthesia with minimal toxicity. Gold nanorods (GNRs) that can convert near-infrared (NIR) light into heat were attached to liposomes (Lip-GNRs), enabling light-triggered phase transition of their lipid bilayers with a consequent release of payload. Lip-GNRs containing the site 1 sodium channel blocker tetrodotoxin and the α2-adrenergic agonist dexmedetomidine (Lip-GNR-TD) were injected subcutaneously in the rat footpad. Irradiation with an 808 nm continuous wave NIR laser produced on-demand and repeated infiltration anesthesia in the rat footpad in proportion to the irradiance, with minimal toxicity. The ability to achieve on-demand and repeated local anesthesia could be very beneficial in the management of pain.

Amphetamine elevates nucleus accumbens dopamine via an action potential-dependent mechanism that is modulated by endocannabinoids.

The reinforcing effects of abused drugs are mediated by their ability to elevate nucleus accumbens dopamine. Amphetamine (AMPH) was historically thought to increase dopamine by an action potential-independent, non-exocytotic type of release called efflux, involving reversal of dopamine transporter function and driven by vesicular dopamine depletion. Growing evidence suggests that AMPH also acts by an action potential-dependent mechanism. Indeed, fast-scan cyclic voltammetry demonstrates that AMPH activates dopamine transients, reward-related phasic signals generated by burst firing of dopamine neurons and dependent on intact vesicular dopamine. Not established for AMPH but indicating a shared mechanism, endocannabinoids facilitate this activation of dopamine transients by broad classes of abused drugs. Here, using fast-scan cyclic voltammetry coupled to pharmacological manipulations in awake rats, we investigated the action potential and endocannabinoid dependence of AMPH-induced elevations in nucleus accumbens dopamine. AMPH increased the frequency, amplitude and duration of transients, which were observed riding on top of slower dopamine increases. Surprisingly, silencing dopamine neuron firing abolished all AMPH-induced dopamine elevations, identifying an action potential-dependent origin. Blocking cannabinoid type 1 receptors prevented AMPH from increasing transient frequency, similar to reported effects on other abused drugs, but not from increasing transient duration and inhibiting dopamine uptake. Thus, AMPH elevates nucleus accumbens dopamine by eliciting transients via cannabinoid type 1 receptors and promoting the summation of temporally coincident transients, made more numerous, larger and wider by AMPH. Collectively, these findings are inconsistent with AMPH eliciting action potential-independent dopamine efflux and vesicular dopamine depletion, and support endocannabinoids facilitating phasic dopamine signalling as a common action in drug reinforcement.

Tetrodotoxin blockade on canine cardiac L-type Ca²âº channels depends on pH and redox potential.

Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na⁺ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca²âº current (I(Ca)) in canine cardiac cells. In the present study, the TTX-sensitivity of I(Ca) was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC50 value obtained under physiological conditions) caused 60% ± 2% inhibition of I(Ca) in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 µM H2O2). Phosphorylation of the channel protein (induced by 3 µM forskolin) failed to modify the inhibiting potency of TTX; an IC50 value of 50 ± 4 µM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels.

Interhemispheric regulation of the medial prefrontal cortical glutamate stress response in rats.

While stressors are known to increase medial prefrontal cortex (PFC) glutamate (GLU) levels, the mechanism(s) subserving this response remain to be elucidated. We used microdialysis and local drug applications to investigate, in male Long-Evans rats, whether the PFC GLU stress response might reflect increased interhemispheric communication by callosal projection neurons. We report here that tail-pinch stress (20 min) elicited comparable increases in GLU in the left and right PFC that were sodium and calcium dependent and insensitive to local glial cystine-GLU exchanger blockade. Unilateral ibotenate-induced PFC lesions abolished the GLU stress response in the opposite hemisphere, as did contralateral mGlu(2/3) receptor activation. Local dopamine (DA) D(1) receptor blockade in the left PFC potently enhanced the right PFC GLU stress response, whereas the same treatment applied to the right PFC had a much weaker effect on the left PFC GLU response. Finally, the PFC GLU stress response was attenuated and potentiated, respectively, following alpha(1)-adrenoreceptor blockade and GABA(B) receptor activation in the opposite hemisphere. These findings indicate that the PFC GLU stress response reflects, at least in part, activation of callosal neurons located in the opposite hemisphere and that stress-induced activation of these neurons is regulated by GLU-, DA-, norepinephrine-, and GABA-sensitive mechanisms. In the case of DA, this control is asymmetrical, with a marked regulatory bias of the left PFC DA input over the right PFC GLU stress response. Together, these findings suggest that callosal neurons and their afferentation play an important role in the hemispheric specialization of PFC-mediated responses to stressors.

Reducing the late sodium current improves cardiac function during sodium pump inhibition by ouabain.

Inhibition by cardiac glycosides of Na(+), K(+)-ATPase reduces sodium efflux from myocytes and may lead to Na(+) and Ca(2+) overload and detrimental effects on mechanical function, energy metabolism, and electrical activity. We hypothesized that inhibition of sodium persistent inward current (late I(Na)) would reduce ouabain's effect to cause cellular Na(+) loading and its detrimental metabolic (decrease of ATP) and functional (arrhythmias, contracture) effects. Therefore, we determined effects of ouabain on concentrations of intracellular sodium (Na(+)(i)) and high-energy phosphates using (23)Na and (31)P NMR, the amplitude of late I(Na) using the whole-cell patch-clamp technique, and contractility and electrical activity of guinea pig isolated hearts, papillary muscles, and ventricular myocytes in the absence and presence of inhibitors of late I(Na). Ouabain (1-1.3 µM) increased Na(+)(i) and late I(Na) of guinea pig isolated hearts and myocytes by 3.7- and 4.2-fold, respectively. The late I(Na) inhibitors ranolazine and tetrodotoxin significantly reduced ouabain-stimulated increases in Na(+)(i) and late I(Na). Reductions of ATP and phosphocreatine contents and increased diastolic tension in ouabain-treated hearts were also markedly attenuated by ranolazine. Furthermore, the ouabain-induced increase of late I(Na) was also attenuated by the Ca(2+)-calmodulin-dependent kinase I inhibitors KN-93 [N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide] and autocamide-2 related inhibitory peptide, but not by KN-92 [2-[N-(4'-methoxybenzenesulfonyl)]amino-N-(4'-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate]. We conclude that ouabain-induced Na(+) and Ca(2+) overload is ameliorated by the inhibition of late I(Na).

Exploring the mechanisms of intravesical electrical stimulation in the in vitro rat whole bladder after treatment with atropine, α,ß-methylATP and tetrodotoxin.

AIMS: In a previous study, we showed that the working mechanism of intravesical electrical stimulation (IVES) is probably mainly nerve mediated. But even after bladder decentralization, IVES can induce detrusor contraction. This study explores the effect of IVES in decentralized bladders and the importance of receptors in the bladder wall for a response on IVES. METHODS: IVES (10 Hz square wave pulses, 20 msec pulse duration, 6 mA) was used in the bladder of 16 female Sprague-Dawley rats. After repeating IVES after consecutive bilateral bladder nerves section (L6-roots, pelvic nerves, and major pelvic ganglion (MPG)), the bladders were mounted in a tissue bath. IVES was performed in the control (n=16), after administration of tetrodotoxin (TTX) (n=6), after atropine and atropine with α,ß-methylATP (n=6), and after α,ß-methylATP and α,ß-methylATP with atropine (n=4). The IVES-induced pressure rise (ΔP) was recorded. RESULTS: Maximum ΔP (maxΔP) after transection of the MPG was significantly lower than after pelvic nerves transection. Treatment with TTX and with α,ß-methylATP plus atropine abolished ΔP. Atropine alone gave an insignificant decrease of maxΔP. Treatment with α,ß-methylATP alone reduced maxΔP significantly. CONCLUSIONS: IVES can evoke contractions in a decentralized bladder. IVES-induced contractions are not a result of direct muscle stimulation, but are nerve mediated, involving intramural innervation and several parts of the bladder innervation. IVES-evoked contraction can be divided in a, contraction duration determining, cholinergic part and a, contraction strength determining, purinergic part. The peripheral innervation could play a role in IVES treatment in patients with interrupted central reflex pathway.

Gender- and age-related differences in muscular and nerve-mediated responses in human colon.

BACKGROUND: Gender- and age-related differences in muscular and nerve-mediated responses in human colon are poorly characterized. We studied carbachol-induced motor responses and electrically evoked contractions in sigmoid circular muscle from adult and elderly patients of different gender. METHODS: Sigmoid colon segments were obtained from 24 men and 16 women undergoing left hemicolectomy for colon cancer. Isometric tension was measured on muscle strips exposed to increasing carbachol concentrations. The effects of atropine, guanethidine, L-nitro arginine methyl ester (L-NAME), and tetrodotoxin on electrically evoked contractions were also studied. RESULTS: Female patients showed higher maximal response to carbachol than male patients, elderly females being the most sensitive to carbachol among all patient groups. Electrically evoked contractions were linearly related to stimulation frequency and abolished by tetrodotoxin. Electrically evoked contractions were significantly more pronounced in elderly male patients; they were reduced by atropine and guanethidine and increased by L-nitro arginine methyl ester in the presence of atropine and guanethidine (P < 0.05). The effect of L-NAME was most marked in elderly male patients and least pronounced in elderly females. CONCLUSIONS: The response to carbachol and the role of nitrergic pathways differ according to age and gender; this may depend on muscarinic receptor upregulation or humoral factors affecting nitric oxide release, respectively.