Skeletal muscle mitoflashes, pH, and the role of uncoupling protein-3.

Mitochondrial reactive oxygen species (ROS) are important cellular signaling molecules, but can cause oxidative damage if not kept within tolerable limits. An important proximal form of ROS in mitochondria is superoxide. Its production is thought to occur in regulated stochastic bursts, but current methods using mitochondrial targeted cpYFP to assess superoxide flashes are confounded by changes in pH. Accordingly, these flashes are generally referred to as 'mitoflashes'. Here we provide regulatory insights into mitoflashes and pH fluctuations in skeletal muscle, and the role of uncoupling protein-3 (UCP3). Using quantitative confocal microscopy of mitoflashes in intact muscle fibers, we show that the mitoflash magnitude significantly correlates with the degree of mitochondrial inner membrane depolarization and ablation of UCP3 did not affect this correlation. We assessed the effects of the absence of UCP3 on mitoflash activity in intact skeletal muscle fibers, and found no effects on mitoflash frequency, amplitude or duration, with a slight reduction in the average size of mitoflashes. We further investigated the regulation of pH flashes (pHlashes, presumably a component of mitoflash) by UCP3 using mitochondrial targeted SypHer (mt-SypHer) in skeletal muscle fibers. The frequency of pHlashes was significantly reduced in the absence of UCP3, without changes in other flash properties. ROS scavenger, tiron, did not alter pHlash frequency in either WT or UCP3KO mice. High resolution respirometry revealed that in the absence of UCP3 there is impaired proton leak and Complex I-driven respiration and maximal coupled respiration. Total cellular production of hydrogen peroxide (H2O2) as detected by Amplex-UltraRed was unaffected. Altogether, we demonstrate a correlation between mitochondrial membrane potential and mitoflash magnitude in skeletal muscle fibers that is independent of UCP3, and a role for UCP3 in the control of pHlash frequency and of proton leak- and Complex I coupled-respiration in skeletal muscle fibers. The differential regulation of mitoflashes and pHlashes by UCP3 and tiron also indicate that the two events, though may be related, are not identical events.

Purinergic Signaling Modulates Survival/Proliferation of Human Dental Pulp Stem Cells.

Human dental pulp stem cells (hDPSCs) reside in postnatal dental pulp and exhibit the potential to differentiate into odontoblasts as well as neurons. However, the intercellular signaling niches necessary for hDPSC survival and self-renewal remain largely unknown. The objective of this study is to demonstrate the existence of intercellular purinergic signaling in hDPSCs and to assess the impact of purinergic signaling on hDPSC survival and proliferation. hDPSCs were isolated from extracted third molars and cultured in minimum essential medium. To demonstrate responsiveness to ATP application and inhibitions by purinergic receptor antagonists, whole cell patch-clamp recordings of ATP-induced currents were recorded from cultured hDPSCs. Immunofluorescence and enzymatic histochemistry staining were performed to assess purinergic receptor expression and ectonucleotidase activity in hDPSCs, respectively. To determine the effects of purinergic signaling on hDPSC, purinergic receptor antagonists and an ectonucleotidase inhibitor were applied in culture medium, and hDPSC survival and proliferation were assessed with DAPI staining and Ki67 immunofluorescence staining, respectively. We demonstrated that ATP application induced inward currents in hDPSCs. P2X and P2Y receptors are involved in the generation of ATP-induced inward currents. We also detected expression of NTPDase3 and ectonucleotidase activity in hDPSCs. We further demonstrated that purinergic receptors were tonically activated in hDPSCs and that inhibition of ectonucleotidase activity enhanced ATP-induced inward currents. Furthermore, we found that blocking P2Y and P2X receptors reduced-and inhibition of ecto-ATPase activity enhanced-the survival and proliferation of hDPSCs, while blocking P2X receptors alone affected only hDPSC proliferation. Autocrine/paracrine purinergic signaling is essential for hDPSC survival and proliferation. These results reveal potential targets to manipulate hDPSCs to promote tooth/dental pulp repair and regeneration.

Primary structure and functional expression of the omega-conotoxin-sensitive N-type calcium channel from rabbit brain.

The complete amino acid sequence of a rabbit brain calcium channel (BIII) has been deduced by cloning and sequencing the cDNA. The open reading frame encodes 2339 amino acids, which corresponds to an M(r) of 261,167. A phylogenetic tree representing evolutionary relationships indicates that BIII is grouped together with the other rabbit brain calcium channels, BI and BII, into a subfamily that is distinct from the dihydropyridine-sensitive L-type subfamily. Transient expression in cultured skeletal muscle myotubes derived from muscular dysgenic mice demonstrates that the BIII channel mediates an omega-conotoxin-sensitive calcium current with kinetics and voltage dependence like those previously reported for whole-cell N-type current. Cell-attached patch recordings, with isotonic barium as the charge carrier, revealed distinct single channels with an average slope conductance of 14.3 pS.

CD73 Controls Extracellular Adenosine Generation in the Trigeminal Nociceptive Nerves.

Purinergic signaling is involved in pain generation and modulation in the nociceptive sensory nervous system. Adenosine triphosphate (ATP) induces pain via activation of ionotropic P2X receptors while adenosine mediates analgesia via activation of metabotropic P1 receptors. These purinergic signaling are determined by ecto-nucleotidases that control ATP degradation and adenosine generation. Using enzymatic histochemistry, we detected ecto-AMPase activity in dental pulp, trigeminal ganglia (TG) neurons, and their nerve fibers. Using immunofluorescence staining, we confirmed the expression of ecto-5'-nucleotidase (CD73) in trigeminal nociceptive neurons and their axonal fibers, including the nociceptive nerve fibers projecting into the brainstem. In addition, we detected the existence of CD73 and ecto-AMPase activity in the nociceptive lamina of the trigeminal subnucleus caudalis (TSNC) in the brainstem. Furthermore, we demonstrated that incubation with specific anti-CD73 serum significantly reduced the ecto-AMPase activity in the nociceptive lamina in the brainstem. Our results indicate that CD73 might participate in nociceptive modulation by affecting extracellular adenosine generation in the trigeminal nociceptive pathway. Disruption of TG neuronal ecto-nucleotidase expression and axonal terminal localization under certain circumstances such as chronic inflammation, oxidant stress, local constriction, and injury in trigeminal nerves may contribute to the pathogenesis of orofacial neuropathic pain.

Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor.

Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca(2)+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH(2)-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca(2)+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca(2)+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 microM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca(2)+ release was smaller and activated at more negative potentials for myotubes expressing the NH(2)-terminal CCD mutations. The shift in the voltage dependence of SR Ca(2)+ release correlated strongly with changes in resting Ca(2)+, SR Ca(2)+ store depletion, and peak voltage-gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca(2)+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca(2)+ leak. These results demonstrate that the NH(2)-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca(2)+ leak, store depletion, and a reduction in voltage-gated Ca(2)+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.

Unitary behavior of skeletal, cardiac, and chimeric L-type Ca2+ channels expressed in dysgenic myotubes.

Skeletal and cardiac dihydropyridine receptors function both as voltage-dependent L-type calcium channels (L-channels) and as critical proteins that trigger calcium release from the sarcoplasmic reticulum in muscle. In spite of these similarities, skeletal L-channels exhibit a markedly slower activation rate than cardiac L-channels. We investigated the mechanisms underlying this difference by comparing the unitary behavior of L-channels in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, or chimeric dihydropyridine receptors. Our results demonstrate that ensemble averages activate rapidly for the purely cardiac dihydropyridine receptor and approximately five times more slowly for L-channels attributable to the purely skeletal dihydropyridine receptor or a chimeric dihydropyridine receptor in which only the first internal repeat and all of the putative intracellular loops are of skeletal origin. All of the constructs studied similarly exhibit a brief (2-ms) and a long (> or = 15-ms) open time in the presence of Bay K 8644, neither of which depend significantly on voltage. In the absence of Bay K 8644, the fraction of total open events is markedly shifted to the briefer open time without altering the rate of ensemble activation. Closed time analysis of L-channels with cardiac-like, rapid activation (recorded in the presence of dihydropyridine agonist) reveals both a brief (approximately 1-ms) closed time and a second, voltage-dependent, long-lasting closed time. The time until first opening after depolarization is three to six times faster for rapidly activating L-channels than for slowly activating L-channels and depends strongly on voltage for both types of channels. The results suggest that a voltage-dependent, closed-closed transition that is fast in cardiac L-channels and slow in skeletal L-channels can account for the difference in activation rate between these two channels.

Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel.

L-type Ca(2+) channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H. T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72-75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q(max), 7.5 +/- 0.8 and 4.5 +/- 0.4 nC/muF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G(max)/Q(max)). Thus, RyR1 enhances both the expression level and Ca(2+) conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A(fast) and A(slow)) and time constants (tau(slow) and tau(fast)) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A(fast)/[A(fast) + A(slow)] approximately 0.25). However, both tau(fast) and tau(slow) were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (tau(fast), 8.5 +/- 1.2 and 4.4 +/- 0.5 ms; tau(slow), 79.5 +/- 10.5 and 34.6 +/- 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 microM) caused a hyperpolarizing shift (approximately 10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A(slow) and A(fast) in normal myotubes, but a large increase in only A(fast) in dyspedic myotubes. Equimolar substitution of Ba(2+) for extracellular Ca(2+) increased both A(fast) and A(slow) in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A(fast) or A(slow). These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca(2+) channel accessory subunit.

Single calcium channel behavior in native skeletal muscle.

The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.

Role of calcium permeation in dihydropyridine receptor function. Insights into channel gating and excitation-contraction coupling.

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809-2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from -20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: tau(act) = 30.7 +/- 1.9 ms, n = 11; CEIIIK: tau(act) = 2.9 +/- 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca(2+) transients with parameters of voltage dependence (V(0.5) = 6.5 +/- 3.2 mV and k = 9.3 +/- 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125-147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitation-contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to -50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 +/- 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Q(off) may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.

External Dentin Stimulation Induces ATP Release in Human Teeth.

ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain.

Pain characteristics help to predict the analgesic efficacy of radiotherapy for the treatment of cancer pain.

It is recognised that radiotherapy provides relief for intractable pain in approximately 50% of patients with cancer pain. Unfortunately, traditional explanatory variables, such as age, gender, histology or radiation dose, do not help to predict which individuals will benefit from palliative radiotherapy. A non-randomised prospective clinical trial was conducted on 51 patients to evaluate the value of pain characteristics as new explanatory variables for predicting the efficacy of palliative radiotherapy for providing cancer pain relief. Two new explanatory variables were identified: the presence of radiating pain and the pain score before radiotherapy.

Effects of the affinity ligands 14-beta-chloroacetylnaltrexone and 14-beta-bromoacetamidomorphine on [3H]-dihydromorphine binding sites in rat brain.

The aim of the present study was to examine the inhibitory effects in vitro of the affinity ligands 14-beta-chloroacetylnaltrexone (CAN) and 14-beta-bromoacetamidomorphine (BAM) to characterize the pharmacological specificity of the ligands for high and low affinity opioid binding sites. Rat brain membranes were incubated with 2.0 microM BAM or CAN, or their parent compounds (morphine and naltrexone, respectively) at 37 degrees for 45 min, and the membranes were washed extensively to remove the unbound ligand. The specific binding of 0.3 nM [3H]dihydromorphine ([3H]DHM) was reduced 32 +/- 7% in membranes treated with CAN and BAM, whereas specific binding in preparations treated with morphine and naltrexone was not significantly different from controls. An increased affinity of BAM and CAN relative to morphine and naltrexone could not account for the observed irreversible inhibition, since the relative affinity of CAN was similar to that of naltrexone and that of BAM was 10-fold less active than morphine. Saturation binding assays revealed that the affinity ligands selectively abolished a high affinity binding site (Kd = 0.3 nM, Bmax 95 fmoles/mg protein), which comprised approximately one-third of the total number of sites. The affinity of the remaining site (Kd = 4.0 nM) was not altered significantly. The results indicate that the inhibition caused by the affinity ligands is irreversible and represents inactivation of high affinity opioid binding sites in a relatively selective manner.

Advanced age alone does not suppress anastomotic healing in the intestine.

BACKGROUND: Because retrospective clinical studies yield conflicting results and experimental data are completely absent, this study was performed to determine whether anastomotic repair in the intestine deteriorates with age. METHODS: Ileal and colonic anastomoses were constructed in two groups of healthy rats, ages 2 to 3 months and 27 to 30 months, respectively. Healing was assessed, both 3 and 7 days after operation, by measuring anastomotic bursting pressure, breaking strength, and collagen content, the latter both biochemically (hydroxyproline) and morphometrically. In addition, the ex vivo collagen synthetic capacities were compared. RESULTS: The development of anastomotic strength was similar in young and old rats; average strength increased from 3 to 7 days and was never lower in the older animals. This was true for both bursting pressure and breaking strength. The collagen production capacity was suppressed in the old rats, particularly in the ileum (p < 0.05), whereas the synthesis of noncollagenous protein remained unaltered. However, this did not result in a reduced accumulation of collagen in the anastomotic area--both anastomotic hydroxyproline content and the volume percentage of collagen in the actual wound area were unchanged. CONCLUSIONS: Advanced age per se does not affect development of strength or deposition of collagen during early repair of intestinal anastomoses.

The relevance of cholinergic transmission at the spinal level to opiate effectiveness.

Rats chronically implanted with intrathecal catheters displayed a dose-dependent increase in the hot-place and tail-flick response latencies following the injection of morphine or nicomorphine into the subarachnoid space through the indwelling catheter. Naloxone inhibited the antinociceptive effect of both opiate drugs, but the inhibition of nicomorphine-induced antinociception was incomplete. To evaluate the importance of cholinergic mechanisms in opiate effectiveness, the interactions with atropine or physostigmine were evaluated. Atropine reduced the effects of morphine and abolished the effects of nicomorphine at the doses used. Physostigmine markedly potentiated morphine effectiveness, but had a negligible effect on nicomorphine effectiveness. It is proposed that these differences relate to a specific cholinergic mechanism involved in antinociception after intrathecal nicomorphine. The data indicate that at a spinal level cholinergic mechanisms are relevant to opiate effectiveness.

Dipropylacetate-induced shaking behaviour in the rat: a role of spinal alpha 2-adrenoceptors.

The role of spinal adrenoceptors in di-n-propylacetate (DPA)-induced shaking behaviour was studied. The alpha 2-agonist p-aminoclonidine in a dose of 5 micrograms was found to suppress the DPA-induced body shakes when injected intrathecally but not when given intraventricularly. There was an enhancement of DPA-induced body shakes after the intrathecal injection of idazoxan while only a slight decrease was found after the intrathecal injection of prazosin. Intrathecal injection of idazoxan but not of prazosin proved to be effective to reverse the DPA-induced body shakes suppressed by p-aminoclonidine. Although the difference in effectiveness after intrathecal and intravenous injection was less than has been described for opiates there are several arguments that indicate an effect confined to the spinal cord. The present results further evidence the notion that the DPA-induced shaking behaviour shares common mechanisms with some of the morphine abstinence symptoms. The data indicate that spinal alpha 2-adrenoceptors are at least partly involved in the suppressive effect of p-aminoclonidine on DPA-induced shaking behaviour.

Antinociceptive effects of intrathecally injected cholinomimetic drugs in the rat and cat.

Rats chronically implanted with intrathecal catheters displayed a dose-dependent increase in the hot-plate, tail-flick response latencies, and decreased the magnitude of the writhing response following the injection of certain cholinomimetics into the subarachnoid space through the indwelling catheter. The structure-activity relationship for these agents is oxotremorine greater than carbachol greater than acetylcholine + physostigmine much much greater than acetylcholine = nicotine-HCl = 0. Atropine, but not naloxone, strychnine, picrotoxin, curare or methysergide and phentolamine, reversed the antinociceptive effect. This suggests the involvement of muscarinic cholinergic mechanisms. Experiments with intrathecal injection of carbachol into the spinal subarachnoid space of cats fitted with intrathecal catheters also revealed a potent antinociceptive effect which was completely antagonized by atropine. The effect was somatotopically limited with the skin surfaces innervated by cord segments nearest the catheter tip showing the most significant effect with the shortest latency of onset. This observation, together with the absence of changes in general reflex motor function or postural control further indicated a selective spinal effectiveness of muscarinic agonists after low dose intrathecal administration.

Autonomic modulation of action potential and tension in guinea pig papillary muscles.

The effects of alpha 1-adrenoceptor and muscarinic acetylcholine receptor stimulation on action potential and tension were studied in guinea pig papillary muscles obtained from both right and left ventricles. Stimulation of muscarinic acetylcholine receptors with carbachol produced a reduction of the action potential duration and a positive inotropic effect in papillary muscles from both ventricles. Both effects were concentration dependent and atropine sensitive. However, differential responsiveness was found upon alpha 1-adrenoceptor activation in muscles obtained from left and right ventricles. In right side papillary muscles, the alpha 1-adrenoceptor agonist, methoxamine, decreased the action potential duration and produced a positive inotropic effect. In contrast, methoxamine decreased the action potential duration but failed to produce a positive inotropic effect in left side papillary muscles. All methoxamine effects were antagonized by prazosin. Responses to maximum concentration of carbachol and methoxamine on the action potential duration and contractility were additive in right side papillary muscles. Phorbol 12,13-dibutyrate (PDB), a direct protein kinase C activator, also decreased the action potential duration in a manner that was additive to both carbachol and methoxamine. However, PDB reversed the positive inotropic effect of carbachol and methoxamine. The methoxamine-induced shortening of the action potential duration was prevented by pretreatment with indomethacin and nordihydroguaiaretic acid, blockers of arachidonic acid metabolism, but not by the protein kinase C antagonist, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7).(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of chronic diazepam effects on the auditory evoked potential of the rat.

The time course of chronic diazepam effects on auditory evoked potentials was studied in rats. Auditory evoked potentials were elicited by background and target tones in a passive oddball paradigm. Diazepam was administered by slow release implants to establish constant blood concentrations. Recordings were made during 21 days of treatment and 9 days after treatment ceased. Diazepam increased the amplitude of the P40 component and decreased the amplitude of the P72-P102 components elicited by background tones. Diazepam increased the amplitude of the P40-P48 component and decreased that of the N58 component elicited by target tones. These effects remained constant during treatment. Diazepam further decreased the amplitude of the P102 component elicited by target tones. This effect became more distinct over time. No group differences were found 9 days after treatment. The constant drug effects on middle-latency components (P40-P48) might reflect diazepam-induced changes in sensory information processing. The decreased long-latency component (P102) might reflect a diminished attention to, or discrimination of, target tones. The time course of this effect might reflect diazepam-enhanced habituation.

A molecular model for the synergic interaction between gamma-aminobutyric acid and general anaesthetics.

Within the context of the discussion about rational polytherapy, we determined the effects of four anaesthetics on the binding of [3H]t-butylbicycloorthobenzoate ([3H]TBOB) to the GABA(A) receptor complex in the presence of several concentrations of GABA (gamma-aminobutyric acid), in order to build a molecular model that can describe and quantify the interactions between the compounds. The empirical isobole method revealed that GABA and the anaesthetics acted synergically in displacing [3H]TBOB. This synergy could be described by a simple molecular model in which both GABA and the anaesthetics displaced [3H]TBOB allosterically and in which GABA allosterically enhanced the binding of the anaesthetics. To get information about the interaction between GABA and anaesthetics, we used [3H]TBOB as a tracer ligand. The model indicated that GABA enhanced the affinity of thiopental 3.0-fold, propofol 5.0-fold, the neuroactive steroids Org 20599 3.5-fold and Org 20549 13-fold. Insight into the molecular mechanism and strength of these interactions can help clinicians to choose therapeutically optimal drug and dose combinations: a step towards rational polytherapy.