Roberto T. Sudo

Dantrolene sodium can increase or attenuate activity of skeletal muscle ryanodine receptor calcium release channel : Clinical implications

Background Dantrolene sodium (DS) is a direct‐acting skeletal muscle relaxant whose only known action is to block calcium release from intracellular storage sites. The exact site of action for DS is unknown, but its efficacy in treating and preventing anesthetic‐induced malignant hyperthermia (MH) is well established. Methods Single ryanodine (Ry1) receptor calcium release channels were incorporated into a planar lipid bilayer for electrophysiologic recording and for subsequent analysis of the channels gating and conductance properties. The cellular effects of low DS concentrations were investigated by isometric contracture tension responses in biopsied MH human and dog muscle fascicles and in normal, single fibers from human vastus lateralis muscle. Results Two concentration‐dependent DS effects on the isolated Ry sub 1 receptor were discovered, suggesting at least two different binding sites. At nanomolar concentrations, DS activated the channel by causing three‐ to fivefold increases in open‐state probability and dwell times. At micromolar concentrations, DS first increased then reduced activity in the channels; with the dominant effect being reduced activity. A 20 nM concentration of DS produced significant contracture tension in human muscle from one MH subject and caused potentiation of twitch in muscle from another MH patient. Halothane contracture in MH dog muscle was followed by an additional increase in tension when treated with 20 nM DS. Other investigations on chemically skinned, human fibers showed that calcium loaded in the sarcoplasmic reticulum was partially released by nM DS. Conclusions The study results suggest that at least two binding sites for DS exist on the Ry1 receptor calcium channel. A low‐affinity (micro Meter) site is associated with reduced channel gating and open‐state dwell time and may relate to the established pharmacologic muscle relaxant effect of DS. The proposed high‐affinity (nM) DS binding site activates the channel, producing Calcium2+ release to the myoplasm, which, under environmentally adverse conditions, could damage genetically predisposed MH muscle. Such a phenomenon, if it occurs in DS‐treated MH patients, could generate a recrudescence of the syndrome.

Studies towards the identification of putative bioactive conformation of potent vasodilator arylidene N-acylhydrazone derivatives

In this report we disclose the synthesis, vasodilatory activity, and identification of bioactive conformation of new N-acylhydrazone and N-methyl-N-acylhydrazone derivatives, structurally designed by bioisosteric replacements of previously described cardioactive compounds LASSBio-294 and its N-methyl derivative LASSBio-785. Some of these novel derivatives presented improved vasorelaxant properties, being new cardiovascular drug candidates.

Is Comparative Cardiotoxicity of S(−) and R(+) Bupivacaine Related to Enantiomer-selective Inhibition of L-type Ca2+ Channels?

Cardiac toxicity can occur after accidental intravascular injection of bupivacaine. Racemic bupivacaine can inhibit both cardiac Na+ and Ca2+ channels, but the contribution of these actions to cardiac depression is not totally understood. We tested whether the effect of R(+) bupivacaine on cardiac electrical activity in isolated hearts and on L-type Ca2+ channels (ICa-L) in isolated cardiac myocytes could be responsible for its increased cardiotoxicity compared with S(−) bupivacaine. Cardiac electrical activity of spontaneously beating isolated hearts was recorded before and after exposure to increasing concentrations of R(+) and S(−) bupivacaine. An increase of the PR interval (80%) and the QRS duration (370%) by 10&mgr;M R(+) bupivacaine (80% and 370%) was significantly higher than for S(−) bupivacaine (25% and 200%, respectively). R(+) but not S(−) bupivacaine produced severe arrhythmias at concentrations larger than 2.5&mgr;M. The intensity of ICa-L inhibition did not differ between bupivacaine isomers. At 0 mV, ICa-L was irreversibly reduced by 40.2% ± 8.8% and 51.4% ± 3.8% in the presence of 10&mgr;M R(+) and S(−) bupivacaine, respectively. The arrhythmogenic effect of R(+) bupivacaine could not be explained by stereoselectivity on the ICa-L inhibition. Thus, other mechanisms could contribute to the cardiac electrical and contractile dysfunction induced by R(+) bupivacaine. Implications Accidental intravascular injection of bupivacaine can induce toxic effects on the heart. We investigated the sensitivity of different bupivacaine structures’ actions on the Ca2+ conducting channels in rat ventricular cells and concluded that the increased toxicity of R(+) bupivacaine is not explained by actions on the Ca2+ channels’ inhibition.

The new compound, LASSBio 294, increases the contractility of intact and saponin‐skinned cardiac muscle from Wistar rats

A new compound designated as LASSBio 294 (L‐294), 3,4‐methylenedioxybenzoyl‐2‐thienylhydrazone, was synthesized as an alternative therapeutic for cardiac dysfunction. L‐294 increased in a dose‐dependent manner the spontaneous contractions of isolated hearts from Wistar rats with maximal effect (128.0±0.7% of control) observed at 25 μM. The positive inotropic effect of L‐294 was also observed in electrically stimulated cardiac tissues from Wistar rats. The maximal increment of twitches, at 200 μM, was 163.1±18.4% for atrial, 153.5±28.5% for papillary and 201.5±18.5% for ventricular muscles. In saponin skinned ventricular cells: (a) L‐294 present in the period of sarcoplasmic reticulum (SR) loading with Ca2+ shifted the dose and caffeine‐induced contracture curve; (b) L‐294 (100 μM) increased 40% the Ca2+ uptake into SR; (c) L‐294 did not significantly alter the sensitivity of contractile proteins to Ca2+ in SR‐disrupted skinned ventricular cells. Retrograde perfusion of the isolated heart from Wistar rats with L‐294 (100 μM) did not cause any significant change in rhythm, heart rate (control, 220±14.7 b.p.m.; 246±24.6 b.p.m. for L‐294), PR interval (control, 66.0±2.4 ms; 64.0±2.3 ms for L‐294) or QRS duration (control, 28.8±3.4 ms; 32.0±2.0 ms for L‐294). These results suggest a novel mechanism for a positive cardioinotropic effect through an interaction with the Ca2+ uptake/release process of the SR. The effect of L‐294 could be explained by a pronounced increased accumulation of Ca2+ into the SR.

Changes in angiotensin receptors expression play a pivotal role in the renal damage observed in spontaneously hypertensive rats

The renal renin-angiotensin system plays a central role in the development of hypertension. The aim of this work was to verify the expression of angiotensin II receptors AT(1)R and AT(2)R in the microsomal fraction of renal cortex and correlate this with the development of hypertension and renal damage in spontaneously hypertensive rats (SHR) using Wistar-Kyoto rats (WKY) as controls. AT(1)R expression increased (126%) and AT(2)R expression decreased (66%) in 4-wk-old SHR; AT(2) expression decreased in 14-wk-old SHR (61%) compared with respective age-matched WKY. These modifications were correlated to the increase in protein kinase C activity and decrease in protein kinase A activity. Four-week-old SHR showed large accumulations of macrophages in kidney glomerulus and the tubulointerstitial area, dense cortical collagen deposition, and arterial proliferative changes in the walls of arterioles and medium-sized vessels. Similar modifications were also observed in 14-wk-old SHR. Four-week-old SHR treated with losartan (30 mg·kg(-1)·day(-1)) or hydralazine (15 and 30 mg·kg(-1)·day(-1)) by gavage for 10 wk did not develop hypertension. The decrease in AT(2)R expression and renal damage observed in SHR remained even after treatment with hydralazine. On the other hand, losartan treatment prevented the modifications observed in 14-wk-old SHR, indicating that renal injuries are caused specifically by AT(1) rather than an increase in blood pressure. Our results indicate that the imbalance in AT(1)R and AT(2)R expression is associated with an inflammatory process that contributes to renal injury in adult SHR and to the development of hypertension.

Na(+)-ATPase in spontaneous hypertensive rats: possible AT(1) receptor target in the development of hypertension.

Clinical and experimental data show an increase in sodium reabsorption on the proximal tubule (PT) in essential hypertension. It is well known that there is a link between essential hypertension and renal angiotensin II (Ang II). The present study was designed to examine ouabain-insensitive Na(+)-ATPase activity and its regulation by Ang II in spontaneously hypertensive rats (SHR). We observed that Na(+)-ATPase activity was enhanced in 14-week-old but not in 6-week-old SHR. The addition of Ang II from 10(-12) to 10(-6) mol/L decreased the enzyme activity in SHR to a level similar to that obtained in WKY. The Ang II inhibitory effect was completely reversed by a specific antagonist of AT(2) receptor, PD123319 (10(-8) mol/L) indicating that a system leading to activation of the enzyme in SHR is inhibited by AT(2)-mediated Ang II. Treatment of SHR with losartan for 10 weeks (weeks 4-14) prevents the increase in Na(+)-ATPase activity observed in 14-week-old SHR. These results indicate a correlation between AT(1) receptor activation in SHR and increased ouabain-insensitive Na(+)-ATPase activity. Our results open new possibilities towards our understanding of the pathophysiological mechanisms involved in the increased sodium reabsorption in PT found in essential hypertension.

The synergistic interaction between morphine and maprotiline after intrathecal injection in rats.

BACKGROUND: Antidepressant drugs act as potent inhibitors of norepinephrine and/or serotonin reuptake and are widely used with opioids for the treatment of chronic pain. The mechanism of this increased analgesic action is unclear. We compared the antinociceptive effects of the intrathecal administration of morphine with that of a nonselective (amitriptyline) or selective (maprotiline or citalopram) antidepressant drug using the thermal withdrawal test in rats. We also investigated the possible mechanisms involved in the interactions of these drugs. METHODS: Male Wistar rats were anesthetized with sevoflurane and administered morphine and antidepressant drugs, or saline, through intrathecal injection. The antinociceptive effect was evaluated using the thermal withdrawal test before and after drug administration. The time for the withdrawal reaction was expressed as percentage of maximum possible effect (MPE). Animals were also pretreated with yohimbine (a nonselective alpha2-adrenergic antagonist) and naloxone (a nonselective opioid antagonist) for mechanism of action studies. Pharmacologic interaction was evaluated using isobolographic analysis of simultaneous administration of fixed proportions of maprotiline and morphine. RESULTS: Single intrathecal administration of morphine (2 &mgr;g), amitriptiline (125 &mgr;g), citalopram (144 &mgr;g), and maprotiline (1.25 &mgr;g) produced 51.6% ± 8.9%, 10.3% ± 3.2%, 33.8% ± 5.2%, and 48.5% ± 9.2% MPE, respectively. The antinociceptive effect of morphine was increased when combined with amitriptyline (91.3% ± 4.6% MPE) and maprotiline (86.9% ± 9.2% MPE) but not with citalopram (40.6% ± 4.6% MPE). Coadministration of maprotiline increased the antinociceptive duration of morphine by 4-fold (from 120 to 480 min), which was reversed by pretreatment with the α-2 adrenoceptor inhibitor, yohimbine, and the mu-type opioid receptor antagonist, naloxone. Isobolographic analysis demonstrated a synergistic interaction between morphine and maprotiline. CONCLUSIONS: Selective norepinephrine reuptake inhibitors can significantly increase the intensity and duration of morphine antinociceptive activity via both α2-adrenergic and opioid receptors. This interaction was not observed with the selective serotonin inhibitor, citalopram.

Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats.

Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N‐acylhydrazone derivative, 3,4‐dimethoxyphenyl‐N‐methyl‐benzoylhydrazide (LASSBio‐1359), on monocrotaline (MCT)‐induced pulmonary hypertension in rats.

Dexmedetomidine prolongs spinal anaesthesia induced by levobupivacaine 0.5% in guinea-pigs.

Alpha‐2 adrenoceptor agonists have been used in association with local anaesthetic to increase the duration of spinal anaesthesia. Intrathecal administration of clonidine prolonged motor blockade induced by local anaesthetic. Since the affinity of dexmedetomidine (DEX) to alpha‐2 adrenoceptors is eight‐times greater than clonidine, it is expected that DEX could be advantageous in clinical anaesthesia. We investigated the duration of motor nerve block induced by spinal injection of 0.5% levobupivacaine (LVB) associated with intrathecal or intraperitoneal administration of DEX. Seventy‐two guinea‐pigs were randomly divided in 12 groups, which were all treated with intrathecal injection of 50 μL of LVB. DEX was injected intrathecally with LVB in 6 groups or injected intraperitoneally after LVB in another 6 groups. Intrathecal DEX (0.1, 0.2 and 0.4 μg) increased the LVB‐induced motor anaesthesia from 48 (41–66) min to 84.5 (52–91) min (P<0.05), 101.5 (83–115) min (P<0.05) and 105 (97–114) min (P<0.05), respectively. Similarly, intraperitoneal DEX (20 and 40 μg kg−1) increased the motor blockade from 48.5 (33–59) min to 88 (71–114) min (P<0.05) and 114.5 (103–156) min (P<0.05), respectively. Pre‐treatment with yohimbine reduced the duration of motor block from 101.5 (83–115) to 76.5 (68–86) min (P<0.05) or from 114.5 (103–156) to 90 (83–93) min (P<0.05) when DEX was administered by the intrathecal or intraperitoneal routes. Motor block induced by spinal injection of LVB was prolonged by intrathecal and systemic administration of DEX, which was partially dependent on activation of alpha‐2 adrenoceptors.

Microwave-assisted synthesis and structure-activity relationships of neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives.

We described herein the optimization of the synthetic methodology exploited to obtain the pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine sedative prototype 1a and novel analogues designed by successive molecular simplifications. By applying microwave irradiation during the hetero Diels-Alder key-step to obtain the heterotricyclic scaffold, under solvent-free conditions, we were able to obtain the desired compounds in drastically shorter times and better yields. Additionally, in vivo evaluation of the sedative effects of these heterocyclic derivatives showed that 1a and the novel structurally-related analogue 1e were the most efficient compounds to impair the locomotor activity in mice at the dose of 10micromol/kg.