Trimetaphan camsilate 250mg/5ml solution for injection ampoules

Remote ischemic preconditioning (RIPC) is an intrinsic phenomenon whereby 3~4 consecutive ischemia-reperfusion cycles to a remote tissue (noncardiac) increases the tolerance of the myocardium to sustained ischemiareperfusion induced injury. Remote ischemic preconditioning induces the local release of chemical mediators which activate the sensory nerve endings to convey signals to the brain. The latter consequently stimulates the efferent nerve endings innervating the myocardium to induce cardioprotection. Indeed, RIPC-induced cardioprotective effects are reliant on the presence of intact neuronal pathways, which has been confirmed using nerve resection of nerves including femoral nerve, vagus nerve, and sciatic nerve. The involvement of neurogenic signaling has been further substantiated using various pharmacological modulators including hexamethonium and trimetaphan. The present review focuses on the potential involvement of neurogenic pathways in mediating remote ischemic preconditioning-induced cardioprotection.

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BACKGROUND: Penile erection is a complex reflex under spinal control and modulated by the brain. The hemodynamic events under autonomic control and the perineal muscles somatic activity are interconnected during the reflex erection at the spinal level, however if the afferent feedback on the corpus cavernosum pressure during an erection affects the somatic activity (perineal muscles contractions) and vice versa is not known. This study was aimed to test this hypothesis using a rat model. METHODS: Intracavernous pressure (ICP) and bulbocavernosus (BC) muscle EMG were recorded during reflex erections elicited with dorsal penile nerve (DNP) electrical stimulation in anaesthetized acutely spinalized SD rats with surgically (bilateral cavernous nerve section, CnX, n = 8) and pharmacologically (trimetaphan infusion, TMPh, n = 8) abolished pressor response, or with surgically (bilateral section of the motor branch of the pudendal nerve, PnX, n = 7) and pharmacologically (1 mg/kg d-tubocurarine, n = 8) blocked perineal muscles contractions, or with interrupted afferent input from the penis (bilateral crush of the dorsal penile nerve, DPnX, n = 7). Control rats (n = 8) received no intervention. RESULTS: Moderate positive correlations were found between net parameters of pressor and somatic activity during DNP-stimulation induced reflex erection in spinal rats, particularly the speed of pressor response development was positively correlated to EMG parameters. No changes of EMG activity were found in CnX rats, while the decrease of BC EMG in TMPh-treated males can be attributed to direct inhibitory action of TMPh on neuromuscular transmission. Pressor response latency was increased and ICP front slope decreased in dTK and PnX rats, indicating that perineal muscles contraction augment pressor response. DPN crush had little effect on ICP and EMG. CONCLUSION: Afferent input on the level of intracavernous pressure and the perineal muscles activity has minimal impact on, correspondingly, the somatic and the autonomic components of the reflex erection in spinal males, once the reflex has been initiated.

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We compared the main properties of human recombinant α3β4β3 neuronal nicotinic receptors with those of α3β4 receptors, expressed in Xenopus oocytes. β3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (α3β4) to 46.7 picosiemens (α3β4β3; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of β3-containing receptors differed little from those of α3β4. The main pharmacological difference in α3β4β3 “triplet” receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine >> lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2–1 μm) and dihydro-β-erythroidine (30 μm) was similar for the two combinations, with a Schild KB for trimetaphan of 76 and 66 nm on α3β4 and α3β4β3, respectively. The change in single channel conductance confirms that β3 replaces a β4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the β3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of β3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics. We compared the main properties of human recombinant α3β4β3 neuronal nicotinic receptors with those of α3β4 receptors, expressed in Xenopus oocytes. β3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (α3β4) to 46.7 picosiemens (α3β4β3; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of β3-containing receptors differed little from those of α3β4. The main pharmacological difference in α3β4β3 “triplet” receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine >> lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2–1 μm) and dihydro-β-erythroidine (30 μm) was similar for the two combinations, with a Schild KB for trimetaphan of 76 and 66 nm on α3β4 and α3β4β3, respectively. The change in single channel conductance confirms that β3 replaces a β4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the β3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of β3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics. Among neuronal nicotinic subunits, β3 was long considered an “orphan” as it does not form functional recombinant receptors if expressed as a classical heteromeric combination, i.e. together with either an α or a β subunit. Because of the sequence similarity between β3 and α5 we tested the hypothesis that, just like α5, β3 would form functional receptors only if co-expressed together with both an α and a β subunit. By a reporter mutation strategy, we showed that β3 is indeed incorporated into functional recombinant α3β4β3 receptors (1Groot-Kormelink P.J. Luyten W.H.M.L. Colquhoun D. Sivilotti L.G. J. Biol. Chem. 1998; 273: 15317-15320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In α3β4β3 receptors, β3 is present as a single copy, which replaces one of the β subunits (versus two copies each for the α3 and β4 subunits (2Boorman J.P. Groot-Kormelink P.J. Sivilotti L.G. J. Physiol. (Lond.). 2000; 529: 567-578Crossref Scopus (64) Google Scholar)); note that β3 coassembles with other β subunits in rat native cerebellar nAChRs 1The abbreviations used are: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; DMPP 1,1-dimethyl-4-phenylpiperazinium.1The abbreviations used are: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; DMPP 1,1-dimethyl-4-phenylpiperazinium. (3Forsayeth J.R. Kobrin E. J. Neurosci. 1997; 17: 1531-1538Crossref PubMed Google Scholar). The question now arises of whether the β3 subunit can change the properties of neuronal nicotinic receptors. There are several reasons for investigating this problem. First of all it is important to establish whether any such change introduced by the presence of β3 affects the role that these receptors may have in physiological processes in the central nervous system or in the pharmacology of tobacco addiction. Furthermore, if β3-containing receptors have distinctive biophysical or pharmacological properties, such receptors can in principle be recognized in native tissue by functional assays. Finally, clear changes in the receptor pharmacology and particularly in the binding affinity of competitive antagonists would be a strong indication that the binding sites of the receptor have changed and that β3 directly forms one of the two interface binding sites. The shorter openings and bursts together with the increased single-channel conductance observed with the co-expression of β3 confirm that β3 is incorporated into the receptor complex. The single-channel conductance change and the stoichiometry of the receptor (2Boorman J.P. Groot-Kormelink P.J. Sivilotti L.G. J. Physiol. (Lond.). 2000; 529: 567-578Crossref Scopus (64) Google Scholar) suggest that β3 takes the place of a classical β subunit. The calcium permeability of the receptor and its sensitivity to a range of agonists and antagonists were unchanged apart from a decrease in the relative potency of the agonist lobeline. These results suggest that β3 either does not participate in the formation of the agonist binding site or that the sequences of β3 and β4 in the relevant domains are too similar to allow them to be differentiated. The extent and nature of changes introduced into the nAChR by the presence of β3 were not sufficient to provide tools for the identification of such receptors in native tissue. Nevertheless, the shorter burst length of these receptors implies a faster decay of any synaptic current that these channels may mediate and suggests a functional role for the β3 subunit in the diverse family of neuronal nicotinic receptor subunits. Expression of Nicotinic Subunits cRNA in the Xenopus oocyte—cDNAs for the human α3, β3, and β4 (GenBank™ accession numbers Y08418, Y08417, and Y08416, respectively) containing only coding sequences and an added Kozak consensus sequence (GCCACC) immediately upstream of the start codon (4Groot-Kormelink P.J. Luyten W.H.M.L. FEBS Lett. 1997; 400: 309-314Crossref PubMed Scopus (45) Google Scholar) were subcloned into the pSP64GL vector, which contains 5′- and 3′-untranslated Xenopus β-globin regions (5Akopian A.N. Sivilotti L. Wood J.N. Nature. 1996; 379: 257-262Crossref PubMed Scopus (903) Google Scholar). All cDNA/pSP64GL plasmids were linearized immediately downstream of the 3′-untranslated β-globin sequence, and cRNA was transcribed using the SP6 Mmessage Mmachine Kit (Ambion). The quality and quantity of cRNAs were checked by gel electrophoresis and comparison with RNA concentration and size markers. Female Xenopus laevis frogs were anesthetized by immersion in neutralized ethyl m-aminobenzoate solution (tricaine, methane sulfonate salt; 0.2% solution weight/volume) and killed by decapitation and destruction of the brain and spinal cord (in accordance to Home Office guidelines) before removal of the ovarian lobes. Clumps of stage V-VI oocytes were dissected in a sterile modified Barth's solution with a composition of 88 mm NaCl, 1 mm KCl, 0.82 mm MgCl2, 0.77 mm CaCl2, 2.4 mm NaHCO3, 15 mm Tris-HCl in high performance liquid chromatography-grade water with 50 units ml–1 penicillin and 50 μg ml–1 streptomycin (Invitrogen), pH 7.4, adjusted with NaOH. The dissected oocytes were treated with collagenase (type IA, Sigma; 65 min at 18 °C, 245 collagen digestion units ml–1 in Barth's solution, 10–12 oocytes/ml), rinsed, stored at 4 °C overnight, and manually defolliculated the following day before cRNA injection (46 nl/oocyte). The oocytes were incubated for ∼60 h at 18 °C in Barth's solution containing 5% heat-inactivated horse serum (Invitrogen) (6Quick M.W. Lester H.A. Methods Neurosci. 1994; 19: 261-279Crossref Scopus (100) Google Scholar) and then stored at 4 °C. Experiments were carried out at a room temperature of 18–20 °C between 2.5 and 14 days from injection. cRNA was injected at a ratio of 1:1 in order to express α3β4 receptors and at a ratio of 1:1:20 (α3:β4:β3) in order to express α3β4β3 receptors in conditions that minimized the presence of pair α3β4 receptors. We have previously found that with this ratio the proportion of current through α3β4 receptors is too small to be detected by fitting doseresponse curves, since it is 39% when the ratio for α3:β4:β3 is 1:1:1 (1Groot-Kormelink P.J. Luyten W.H.M.L. Colquhoun D. Sivilotti L.G. J. Biol. Chem. 1998; 273: 15317-15320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The total amount of cRNA to be injected (in 46 nl of RNase-free water) for each combination was determined empirically, with the aim of achieving an optimal signal to noise ratio in the different experiments. Given that most of the experiments described here were carried out at low agonist concentrations, the level of expression we tried to achieve was higher than that needed for full agonist dose-response curves. The average quantity of cRNA injected in each oocyte was 20 and 30 ng for α3β4 and α3β4β3, respectively. Single Channel Recording—Recordings were obtained in the cell-attached configuration from oocytes that had been stripped of their vitelline membrane after in solution min with mm 20 mm KCl, 1 mm MgCl2, mm pH 7.4, with were from with and to have a of when with low divalent external solution mm NaCl, mm KCl, mm MgCl2, mm CaCl2, mm pH with which 1 as channel openings other than those to channels were observed in the absence of in the solution J. Physiol. PubMed Scopus Google Scholar). were in high solution mm KCl, mm mm pH with to the membrane potential of the oocyte to a and to were at and at 30 The used for were by were with and of D. to a mean for the of the openings at and the mean from each and for α3β4 and α3β4β3, respectively) were for the different to the in which were with a to slope conductance of D. were from the at a potential of were used for time and by a method with a of and time were with a of approximate correction of the mean open time for missed was carried out as previously D. J. Physiol. (Lond.). Scopus Google Scholar). were as of openings by which were shorter than a We the by time and the of the time of their since the time of between bursts are to on both the agonist concentration and the of channels in a The was on average for α3β4 and for in a were at with modified solution mm NaCl, mm KCl, mm mm MgCl2, pH adjusted with and at using the of an were from and with The was on the Experiments were if the total current to the effect of We a solution to the of this is to the Xenopus oocyte and may be by calcium through the neuronal nicotinic channels J. Full Text PDF PubMed Scopus Google Scholar). from were the for a sufficient to a low or the of a after a the higher the current was on a for of min was between this was found to be sufficient to for in agonist sensitivity the a concentration of for the combination was The was only after that this concentration The average changes in the to this concentration observed by the of the for the different from to All the are for the this not the of the results of the were similar not to each of were obtained following a previously described Sivilotti L.G. Colquhoun D. J. Physiol. (Lond.). 1994; Scopus Google Scholar). The aim of this was to or dose-response at the agonist that an signal to noise was to the size for all the agonists tested in a These conditions were to the of and agonist and that the slope of the dose-response was similar for all the agonists the in and Given the of agonists only a be tested in one was tested in and was used as a All obtained in each were by of with to be are the of fitting with a in which the is to be with to the measured In these the the between the dose-response of the different agonists and that of the were obtained as concentration with to The potency in are the of the concentration and for potency were by D. on Scholar). that the of was were it to of the slope for each agonist The agonist used were for for for cytisine, for 1,1-dimethyl-4-phenylpiperazinium 20 for and and for that at 20 than 5% of its for both potency of lobeline relative to is in receptors containing the β3 subunit. the current to of low of or lobeline to α3β4 or α3β4β3 receptors. from such are in The to ACh, and carbachol to that in the on triplet α3β4β3 receptors the lobeline is to that of than it is in oocytes α3β4 of a of nicotinic agonists on human α3β4 and α3β4β3 ratio ratio between α3β4 and α3β4β3 between α3β4 and α3β4β3 between α3β4 and α3β4β3 in a experiments were carried out to confirm the competitive nature of the and to the by the Schild were obtained at low agonist in solution and then in the presence of an this was present both in the and in the agonist were incubated with the for min before to were one concentration was tested in each oocyte in order to the by be in the presence of the from oocytes in which the agonist after to at that of were in the In a similar to that for the agonist potency the ratio by the was measured for each by fitting to the to be were also there was no difference between the slope of the concentration in conditions and in trimetaphan that the were by the Schild were as a Schild and by a is the concentration of and a is a in order to the Schild slope found to be to 1 we the with the Schild to KB the of the effect of trimetaphan on α3β4 and α3β4β3 in a measured the effect of different calcium on the reversal potential of the current by from to were in the and in the both in conditions and in the presence of the agonist current was were and was using were at at and stored on for The of the oocyte conductance would be by calcium through nicotinic was minimized by the oocytes in low calcium solution for h mm mm 2.5 mm mm mm pH with Experiments were also carried out in modified The composition of the low calcium was mm mm mm 2.5 mm mm pH with the high calcium 18 mm and no was and All were carried out with with 2.5 and mm using a as a following were from acetylcholine or calcium dihydro-β-erythroidine and was from All other were from Single Channel by the cell-attached in and co-expression of the β3 subunit had a effect on the properties of α3β4 neuronal nicotinic receptors. The in the presence of 1 at that the openings of β3-containing receptors were and in shorter bursts also the to the of the The open in the to that a single for the of the open for these two of and for α3β4 and α3β4β3, respectively. the of the open which a open channel a for neuronal nicotinic receptors be native or recombinant Colquhoun D. J. Physiol. (Lond.). Scopus Google L.G. Colquhoun D. J. Physiol. (Lond.). 1997; Scopus Google Scholar). The slope conductance of these channels by fitting in was 28.8 picosiemens for the α3β4 combination and 46.7 picosiemens for α3β4β3 receptors The most pronounced effect of the presence of the β3 subunit in the receptor was on the kinetics of the The of 1 that most of the openings of α3β4 channels in These long bursts were from from α3β4β3 time for for the two subunit β3 incorporation a shorter open to the in the open was for β3-containing receptors of all and for α3β4 of all time were all with a of the 4 for α3β4 and the for were from to and as burst The faster time is shorter in the α3β4 and the of in this be this would in the of α3β4 Given that it is that openings are missed at of we an approximate correction for missed D. J. Physiol. (Lond.). Scopus Google this not change the effect of β3 incorporation open were and in α3β4 and α3β4β3, of time for cell-attached from oocytes α3β4 or open open for missed burst length in a of average burst length the shortening that is from of the mean burst length was and for α3β4 and α3β4β3 receptors, 1 a potential is that there are of in α3β4 We considered the that the of shorter bursts was similar to the α3β4β3 bursts and with β3 this we measured mean burst length in α3β4 after long bursts for was by and was on average in the the average for the bursts was 14 than that of α3β4β3 were no differences between α3β4 and α3β4β3 receptors in the time of agonist or the rank order of potency for an of agonists (epibatidine lobeline > cytisine, nicotine > > rank order of potency is similar to that found for human or rat α3β4 receptors by J. 1997; Google Scholar) and Google Scholar) by 50 in oocytes or Nevertheless, there was a decrease in the potency of lobeline with the incorporation of β3, from to relative to for the α3β4 and α3β4β3 receptors, the different between the dose-response to lobeline in and that to the The for each agonist were similar on the two different and range of slope is in the for the α3β4 receptor from with those observed here Sivilotti L.G. Colquhoun D. J. Physiol. (Lond.). 1994; Scopus Google Scholar) to in of J. 1997; Google Scholar). that the were observed in the presence of low calcium than calcium and Sivilotti L.G. Colquhoun D. J. Physiol. (Lond.). 1994; Scopus Google Scholar). as a competitive on native nAChRs J. Physiol. (Lond.). Scopus Google which are to the α3 and β4 subunits. Schild of the of trimetaphan would be for receptor and binding site are in the neuronal nicotinic and whether the site with the is changed by β3 In both receptor trimetaphan (0.2–1 μm) a in the its competitive of was by the slope of the Schild to The incorporation of the β3 subunits had only a effect on the KB for it from to nm not on receptors, the dihydro-β-erythroidine is to be competitive J. 1998; PubMed Scopus Google Scholar). In with we observed a in the in the presence of 30 with similar for α3β4 and α3β4β3 that there is no change in the binding On is a channel at E. 2000; PubMed Scopus Google Scholar) may at J. Physiol. (Lond.). Scopus Google Scholar). In the concentration needed to a was high μm) and had channel as a in the slope of the the effect was similar for α3β4 and α3β4β3 receptors not was similar for the two The reversal potential after a increase in calcium was and for α3β4 and α3β4β3, the of for α3β4 receptors J. J. 1998; Google of changes in external calcium on the reversal potential of the nicotinic current through α3β4 and α3β4β3 in reversal mm mm in a results that the β3 subunit into neuronal nicotinic α3β4 receptors and changes their single channel properties, shortening open and bursts and single channel On the other hand, the presence of β3 had only small or on the receptor sensitivity to a of nicotinic agonists or to the competitive antagonists trimetaphan and dihydro-β-erythroidine and on the channel permeability to of these changes is to be in the identification of native β3-containing receptors; the decrease in lobeline potency is and the increase in single channel conductance is not neuronal nAChRs a range of with several for each combination L.G. Colquhoun D. J. Physiol. (Lond.). 1997; Scopus Google Scholar). The main difference observed in the single-channel of β3-containing receptors was a decrease in the of open and burst length. In an i.e. with the of open only the of the and the properties of the The open measured from a by as In it is that we missed only we an approximate correction for missed to the open not change the effect of β3 important of the decrease in burst length we observed is that synaptic currents mediated by such channels would decay faster than those mediated by α3β4 is that long bursts may be a only of recombinant α3β4 receptors, particularly in the oocyte expression system L.G. Colquhoun D. J. Physiol. (Lond.). 1997; Scopus Google Sivilotti L.G. Colquhoun D. J. Physiol. (Lond.). 1997; Scopus Google Scholar). On the other hand, channel openings similar to recombinant long bursts have been from the which α3 and β4 subunits Colquhoun D. J. Physiol. (Lond.). Scopus Google it is that incorporation of the β3 subunit does this effect in native of the role of the β3 subunit in the receptor showed that α3β4β3 receptors two copies each of α3 and β4 and only one of β3 (2Boorman J.P. Groot-Kormelink P.J. Sivilotti L.G. J. Physiol. (Lond.). 2000; 529: 567-578Crossref Scopus (64) Google Scholar). single channel are in with this as the increase in conductance with β3 incorporation that the β3 subunit takes the place of one of the β4 copies in the α3β4 pentamer. In nicotinic receptors conductance is determined by the at each of of at and of the the and of J. Nature. PubMed Scopus Google Scholar). the and all the subunits we expressed in this are similar and a or at the α3 and β3 have a whereas β4 a β3 can only the of if it takes the place of a β4 in this the on the would by two the conductance increase we it been that to in the of one of the copies of is sufficient to increase the conductance of nAChRs by in solution and using as the E. Nature. PubMed Scopus Google Scholar). of the α5 subunit is also to increase single channel conductance of neuronal nAChR L.G. Colquhoun D. J. Physiol. (Lond.). 1997; Scopus Google J. L. Nature. 1996; PubMed Scopus Google J. J. Physiol. (Lond.). Scopus Google by a similar effect on the that α5, not β3, incorporation also by one the on the β3 an α than a β the only change in the important for conductance would be a for in which in nicotinic receptors a small increase in only if is the main J. Nature. PubMed Scopus Google Biol. PubMed Scopus Google Scholar). that β3 replaces a β4 subunit open the question if β3 participate as the or to one of the two agonist binding sites at the subunit changes in the binding sites that the binding was not by β3 from the results of Schild for trimetaphan and changes in the agonist binding may not be by competitive antagonists their binding site does not with that of the agonist also that the range of competitive antagonists is may be the for a β4 to β3 as the sequences of β4 and β3 in and are that the important in these domains are not as as those for that form the of the interface J. Nature. PubMed Scopus Google Scholar). β3 can in principle only change one of the binding sites, not The Schild method the of the affinity as of one site only is sufficient to receptor nAChR with one agonist and one have low and J. Biol. Chem. Full Text PDF PubMed Google any decrease in the site affinity for the would not be from the agonist experiments also against changes in binding a range of nicotinic the only change we observed with β3 incorporation was a decrease in the relative potency of lobeline. In differences in agonist potency can be to changes in either the binding or which be at level D. J. 1998; Scopus Google Scholar). The of open strong of a of the shorter open observed in β3-containing receptors. change to be the most of the of β3 is needed to as we previously in receptors containing β3 there is no change in the of or nicotine and in the to of the agonist nicotine (1Groot-Kormelink P.J. Luyten W.H.M.L. Colquhoun D. Sivilotti L.G. J. Biol. Chem. 1998; 273: 15317-15320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). that the receptor binding sites are not changed by β3 either β3 the role of a subunit or its to the binding site be detected the sequences of the relevant domains are too similar to those of may only be by a different strategy, such as β3 after the of at binding site differences for such for the on the of binding for dihydro-β-erythroidine and by and J. Neurosci. 1996; PubMed Google J. 1996; PubMed Scopus Google 1997; PubMed Scopus Google Scholar).

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