Time-dependent Blockade Research Articles

A novel STAT3 inhibitor, S3I-201, attenuates renal interstitial fibroblast activation and interstitial fibrosis in obstructive nephropathy

Accumulation of both interstitial myofibroblasts and excessive production of extracellular matrix proteins is a common pathway contributing to chronic kidney disease. In a number of tissues, activation of STAT3 (signal transducer and activator of transcription 3) increases expression of multiple profibrotic genes. Here, we examined the effect of a STAT3 inhibitor, S3I-201, on activation of renal interstitial fibroblasts and progression of renal fibrosis. Treatment of cultured rat renal interstitial fibroblasts with S3I-201 inhibited their activation, as evidenced by dose- and time-dependent blockade of alpha-smooth muscle actin and fibronectin expression. In a mouse model of renal interstitial fibrosis induced by unilateral ureteral obstruction, STAT3 was activated, and administration of S3I-201 attenuated both this activation and extracellular matrix protein deposition following injury. S3I-201 reduced infiltration of the injured kidney by inflammatory cells and suppressed the injury-induced expression of fibronectin, alpha-smooth muscle actin, and collagen type-1 proteins, as well as the expression of multiple cytokines. Furthermore, S3I-201 inhibited proliferation and induced apoptosis preferentially in renal interstitial fibroblasts of the obstructed kidney. Thus, our results suggest that increased STAT3 activity mediates activation of renal interstitial fibroblasts and the progression of renal fibrosis. Inhibition of STAT3 signaling with S3I-201 may hold therapeutic potential for fibrotic kidney diseases.

Inhibitory Action of Fluvoxamine on Kv1.5 Currents

The voltage-gated K(+) channel Kv1.5 was expressed in Chinese hamster ovary cells, and its interaction with fluvoxamine was studied using a whole-cell patch-clamp technique. Fluvoxamine reduced Kv1.5 whole-cell currents in a reversible, concentration-dependent manner, with an IC(50) value of 2.0 microM, and a Hill coefficient of 0.7. Fluvoxamine-induced inhibition of Kv1.5 caused a time-dependent blockade without modifying the kinetics of current activation. Fluvoxamine additionally inhibited Kv1.5 in a closed/resting or nonconducting state after deactivation. Inhibition increased steeply between -30 and 0 mV, corresponding to the voltage range for channel opening. Inhibition displayed an additional voltage dependence at voltages greater than 0 mV, consistent with an electrical distance of 0.16. Fluvoxamine slowed the deactivation time course and a tail crossover phenomenon was observed when the tail currents, recorded in the presence and absence of fluvoxamine, were superimposed. Inhibition of Kv1.5 by fluvoxamine was use-dependent. These results suggest that fluvoxamine strongly inhibits Kv1.5 currents and the inhibition of Kv1.5 by fluvoxamine is mixed channel-state dependent.

The effect of voltage- and time-dependent IKur blockade in human atrial fibrillation-induced electrical-remodeling model

The effect of voltage- and time-dependent IKur blockade in human atrial fibrillation-induced electrical-remodeling model

Neutralization of snake venom phospholipase A 2 toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A 2 (PLA 2) toxins. In this work we evaluated the influence of C. sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA 2s (crotoxin from C. durissus terrificus, bothropstoxin-I from B. jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 μM) and all other PLA 2 toxins (1.0 μM) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA 2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 °C) significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80–95%). Higher concentration of the C. sylvestris extract (1:10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA 2 inhibitors.

Effect of fiduxosin, an antagonist selective for alpha(1A)- and alpha(1D)-adrenoceptors, on intraurethral and arterial pressure responses in conscious dogs.

Fiduxosin is an alpha(1)-adrenoceptor antagonist with higher affinity for alpha(1A)-adrenoceptors and for alpha(1D)-adrenoceptors than for alpha(1B)-adrenoceptors. Our hypothesis is that such a compound with higher affinity for subtypes implicated in the control of lower urinary tract function and lower affinity for a subtype implicated in the control of arterial pressure could result in a superior clinical profile for the treatment of lower urinary tract symptoms suggestive of benign prostatic obstruction. The purpose of this study was to evaluate the potency and selectivity of fiduxosin for effects on prostatic intraurethral pressure (IUP) versus mean arterial pressure (MAP) relative to current clinical standards, terazosin and tamsulosin, in conscious dogs. Phenylephrine (PE)-induced increases in IUP and MAP were determined before and at various time points after an oral dose of each antagonist. Hypotensive potency was also determined. All three antagonists caused dose- and time-dependent blockade of the IUP and MAP pressor effects of PE. The IUP ED(50) values of fiduxosin, tamsulosin, and terazosin were 0.24, 0.004, and 0.23 mg/kg p.o., respectively. The corresponding MAP ED(50) values were 1.79, 0.006, and 0.09 mg/kg p.o. The rank order of IUP selectivity (ratio) was fiduxosin (7.5-fold), tamsulosin (1.5-fold), and terazosin (0.4 = 2.5-fold MAP-selective). Tamsulosin and terazosin caused dose-dependent hypotension, whereas no change in arterial pressure was seen after fiduxosin. These data, illustrating a superior selectivity profile of fiduxosin, are consistent with our hypothesis.

Influence of temperature upon effects of crotoxin and gamma-irradiated crotoxin at rat neuromuscular transmission

The influence of temperature upon the effects of crotoxin (CTX), from Crotalus durissus terrificus venom, and gamma-irradiated ( 60Co, 2000 Gy) crotoxin (iCTX) was studied in rat neuromuscular transmission ‘in vitro’. Indirect twitches were evoked in the phrenic-diaphragm preparation by supramaximal strength pulses with a duration of 0.5 ms and frequency of 0.5 Hz. The phospholipase A 2 (PLA 2) enzymatic activity of CTX and iCTX was assayed against phosphadityl choline in Triton X-100. At 27°C, CTX (14 μg/ml) did not affect the amplitude of indirectly evoked twitches. However, at 37°C, CTX induced a time-dependent blockade of the neuromuscular transmission that started at 90 min and was completed within 240 min. iCTX (14 μg/ml) was inneffective on the neuromuscular transmission either at 27 or 37°C. The PLA 2 enzymatic activity of CTX at 37°C was 84 and that at 27°C was 27 μmol fatty acid released/min/mg protein, and that of the iCTX at 37°C was 39 μmol fatty acid released/min/mg protein. Thus, it was concluded that the mechanism of detoxification of CTX by gamma radiation at the neuromuscular level relies on the loss of its PLA 2 enzymatic activity.

Time-dependent block of the slowly activating delayed rectifier K(+) current by chromanol 293B in guinea-pig ventricular cells.

The slowly activating delayed rectifier K(+) current (I(Ks)) was recorded in single myocytes dissociated from guinea-pig ventricles and the mechanism underlying the block of I(Ks) by a chromanol derivative, 293B, was investigated. In the presence of 1 - 100 microM 293B, activation phase of I(Ks) was followed by a slower decay during 10 s depolarizing pulses. Both the rate and extent of the decay were increased in a concentration-dependent manner. The relationship between the concentration of 293B and the block showed a Hill's coefficient of approximately 1. The half-inhibitory concentration was approximately 3.0 microM and did not differ significantly at various membrane potentials from +20 to +80 mV. A mathematical model for the 293B block was constructed on the basis of multiple closed and open states for the I(Ks) channels, and the blocking rate was calculated by fitting the model to the original current traces. The blocking rate constant showed a linear function with the 293B concentration, indicating 1 : 1 binding stoichiometry. At +80 mV the blocking rate was 4x10(4) M(-1) s(-1) and the unblocking rate was 0.2 s(-1). The results indicate that 293B is an open channel blocker with relatively smaller blocking rate than those reported so far for time-dependent blockade of various ionic channels.

Veratridine-treated brain slices: a cellular model for epileptiform activity

This study introduces veratridine-treated brain slices as a new in vitro synaptic-independent model for epileptiform discharge. Studies were performed on the hippocampus in rat brain slices using conventional electrophysiological intracellular recording techniques. Veratridine (0.3 μM) produced a time-dependent blockade of synaptic transmission as indicated by inhibition of the evoked population spike in the region CA1 of the hippocampus. However, in the same slices, intracellularly-evoked single action potentials were converted to epileptiform bursting shortly after exposure to veratridine. Additionally, in the veratridine model, spontaneous epileptiform activity developed after prolonged (more than 45 min) superfusion. The model was utilized to examine the action of two antiepileptic drugs: a sodium channel dependent and a synaptic dependent antiepileptic agents. Therapeutic concentrations of valproic acid (VPA, 10–100 μM) inhibited both evoked and spontaneous bursting induced by veratridine. However, therapeutic concentrations of the synaptic-dependent antiepileptic drug phenobarbital (20–40 μM) failed to inhibit veratridine-induced bursting. These results demonstrate that the veratridine-treated brain slice is a simple and reliable model for studying mechanisms of action and for screening of potential sodium channel-dependent antiepileptic drugs.

Time-dependent blockade of STP and LTP in hippocampal slices following acute stress in mice

The characteristics of short-term potentiation (STP) and long-term potentiation (LTP) in the CA1 region of hippocampal slices were determined at various times following exposure to acute stress produced by restraint and tail-shock in mice. In slices prepared from control animals, theta-burst stimulation resulted in a large increase in evoked field excitatory postsynaptic potentials (EPSPs) amplitude and slope that remained stable at least up to 30 min after stimulation. Slices prepared 1 h after stress exhibited a marked decrease in the extent of both STP and LTP. STP and LTP magnitude were still significantly decreased 24 h after stress exposure and were completely restored to control levels by 48 h. These results provide evidence for a reversible impairment of STP and LTP in CA1 following an acute episode of stress, and suggest that stress activates processes different from those activated by LTP-inducing stimuli.

Stable expression and characterization of the human brain potassium channel Kv2.1: blockade by antipsychotic agents

We have cloned the cDNA encoding the voltage-dependent K + channel Kv2.1 from human brain (hKv2.1). RNase protection and RT-PCR (reverse transcriptase-PCR) experiments reveal abundant Kv2.1 transcripts in human brain with virtually no expression detectable in human heart. hKv2.1 has been stably transfected into a human glioblastoma cell line, and transformed cells display large, slowly activating outward currents. The kinetics, steady-state activation and inactivation parameters, and external tetraethylammonium sensitivity were all similar to those described previously for hKv2.1 channels transiently expressed in Xenopus oocytes or other mammalian cell lines. A number of dopamine receptor antagonist/antipsychotic agents were shown to block hKv2.1. Trifluoperizine, trifluperidol and pimozide produced time-dependent blockade of hKv2.1 with IC 50 values of approx. 1–2 μM. The diphenylbutylpiperidine fluspirilene was shown to be 4–5-fold more potent than the other agents tested inhibiting hKv2.1 current with an IC 50 value of 297 nM. The block produced by fluspirilene was both time- and frequency-dependent. Furthermore, fluspirilene (1 μM) shifted the midpotential of the hKv2.1 steady-state inactivation curve by approx. 15 mV in the hyperpolarizing direction. These results demonstrate the usefulness of this transfection system for the pharmacological characterization of hKv2.1. Fluspirilene proved to be a relatively potent blocker of hKv2.1 and may provide a useful starting point for the development of more potent and selective agents active against this brain K + channel.

Cardiorespiratory and Neuromuscular Effects of Freshwater Cyanophyte Anabena flosaquae in Rats

Anatoxin-a, recognised as a potent toxin warfare agent, is considered as a weapon of mass destruction due to its lethal anticholinesterase activity. The intravenous administration of cell-free extract of Anabena flosaquae UTEX-2383(anatoxin-a) produced a transient vasodepressor response followed by a sustained rise in blood pressure. The vasodepressor effect was potentiated by physostigmine and antagonised by atropine and bilateral vagotomy, suggesting the involvement of cholinergic system. On the contrary, the vasopressor response was antagonised by hexamethonium prazosin and hemicholinium-3, indicating that the toxin stimulates the sympathetic system through the release of catecholamines from nerve endings. Prolonged apnoea with attendant bronchoconstriction was observed corresponding to bradycardia and vasppressor response which remained unaltered by atropine while antagonised by bilateral vagotomy. The extract when administered intra-arterially did not modify the apnoea induced by veratridine; but phenyldiguanide potentiated the bronchoconstriction, indicating the involvement of pulmonary vagal afferents. The extract produced a dose- and time-dependent blockade of indirect muscle twitch recorded from gastrocnemius muscle. The neuromuscular blockade was potentiated by neostigmine but unaltered by DTC.

Fast sodium action potentials are generated in the distal apical dendrites of rat hippocampal CA1 pyramidal cells

Intracellular recording techniques were used to monitor the evoked responses in hippocampal CA1 pyramidal cells following focal stimulation of the proximally and distally located apical dendritic synapses. The local anaesthetic, QX-314, when injected into individual cells, caused a complete time-dependent blockade of the proximal and then the distal action potentials at both threshold and suprathreshold levels of stimulation. This progressive blockade of proximal and distal voltage-dependent sodium channels suggests that sodium action potentials can be generated in the distal apical dendrites of CA1 pyramidal cells.

Reversible phosphorylation of eukaryotic initiation factor 2 alpha in response to endoplasmic reticular signaling.

Agents, such as EGTA, thapsigargin, and ionophore A23187, that mobilize sequestered Ca2+ from the endoplasmic reticulum (ER) or dithiothreitol (DTT) that compromises the oxidizing environment of the organelle, disrupt early protein processing and inhibit translational initiation. Increased phosphorylation of eIF-2 alpha (5-fold) and inhibition of eIF-2B activity (50%) occur in intact GH3 cells exposed to these agents for 15 min (Prostko et al. J. Biol. Chem. 267:16751-16754, 1992). Alterations in eIF-2 alpha phosphorylation and translational activity in response to EGTA were reversed by addition of Ca2+ in excess of chelator while responses to DTT were reversible by washing. Exposure for 3 h to either A23187 or DTT, previously shown to induce transcription-dependent translational recovery, resulted in dephosphorylation of eIF-2 alpha in a manner blocked by actinomycin D. Phosphorylation of eIF-2 alpha in response to A23187 or DTT was not prevented by conventional inhibitors of translation including cycloheximide, pactamycin, puromycin, or verrucarin. Prolonged inhibition of protein synthesis to deplete the ER of substrates for protein processing resulted in increased eIF-2 alpha phosphorylation, decreased eIF-2B activity, and reduced monosome content that were indicative of time-dependent blockade; these inhibitors did not abolish polysomal content. Superphosphorylation of eIF-2 alpha occurred upon exposure of these preparations to either A23187 or DTT. Tunicamycin, an inhibitor of co-translational transfer of core oligosaccharide, provoked rapid phosphorylation of eIF-2 alpha and inhibition of translational initiation whereas sugar analog inhibitors of glycoprotein processing did neither. A flow of processible protein to the ER does not appear to be required for the phosphorylation of eIF-2 alpha in response to ER perturbants. We hypothesize that perturbation of the translocon, rather than suppression of protein processing, initiates the signal emanating from the ER culminating in eIF-2 alpha phosphorylation and translational repression.

Antifertility effect of methoxychlor in female rats: Dose- and time-dependent blockade of pregnancy

Long-term exposure to methoxychlor (MXC), an estrogenic pesticide, produces infertility in rats, and short-term exposure blocks the decidual cell response (DCR). To address the short-term effects of MXC on fertility, the differential effects of MXC dosage and timing of administration (relative to implantation) on several gestational parameters were investigated. When MXC was administered during early pregnancy (Days 1–8), a dose-dependent decline in implantations and uterine weight was seen with no effect on ovarian weight or corpora lutea; MXC reduced serum progesterone at all doses. Preimplantation administration of MXC (Days 1–3 of pregnancy) produced a decline in implantations and uterine weight, while postimplantation dosing (Days 4–8 of pregnancy) increased resporptions to 100%, decreased uterine weight, and reduced serum progesterone without affecting the number of implantations, ovarian weight, or number of corpora lutea. The DCR of pseudopregnancy was inhibited by 500 mg/kg/day MXC when administered either pre- or postimplantation, but 200 mg/kg/day was without effect in either regimen. When hormonally primed, long-term ovariectomized rats were exposed to doses of 500 mg/kg/day, MXC blocked the induced DCR seen in controls. The data show that short-term MXC dosing during early pregnancy produces a dose-related infertility. The blockade of pregnancy by the preimplatation administration of MXC may be mediated by a direct effect on preimplantation uterine development. The fetal resorption seen following postimplantation dosing is considered a manifestation of both reduced serum progesterone and the direct disruption of normal decidual development by MXC.

Caramiphen: a non-opioid antitussive with potent anticonvulsant properties in rats

The effect of the non-opioid antitussive caramiphen was studied in the rat maximal electroshock test. Caramiphen produced a dose- and time-dependent blockade of tonic hindlimb extension and was nearly twice as potent as the prototypical anticovulsant drug diphenylhydantoin. Pretreatment with a subthreshold-effective dose of caramiphen potentiated the anticonvulsant effects of diphenylhydantoin, lowering its ED 50 33-fold. The anticonvulsant effects of caramiphen were not associated with its cholinolytic activity since (a) its anticonvulsant effects were not antagonized by physostigmine and (b) the more potent cholinolytic atropine was only weakly effective against maximal electroshock convulsions when tested at doses 25 times the minimally effective dose of caramiphen. Anticonvulsant effects of caramiphen were associated with minimal behavioral impairment. The results demonstrate that caramiphen is a potent anticonvulsant against generalized convulsion and, like other non-opioid antitussives, will enhance the anticonvulsant properties of diphenylhydantoin. It is suggested that the anticonvulsant effects of caramiphen result from specific binding to brain receptors labelled by the non-opioid antitussive dextromethorphan, and that the interactions with diphenylhydantioin involve allosteric interactions between the different bidning sites.

Low Ba-induced pacemaker current in well-polarized cat papillary muscle.

It has previously been reported that superfusion of normally quiescent mammalian ventricular muscle with low concentrations of Ba (less than 0.3 mM) can induce spontaneous activity with maximum diastolic potentials (MDP) that are similar to the normal resting potential (-80 mV or larger). The mechanism for this activity was studied in cat papillary muscle sucrose-gap preparations under current clamp and voltage-clamp conditions. Hyperpolarizing current pulses decreased or abolished the amplitude of the pacemaker potential in a voltage-dependent manner. When Ba concentration was increased to 2 mM the MDP depolarized by approximately 20 mV. Hyperpolarizing steps under these conditions abolished the diastolic depolarization, also in a voltage-dependent manner. Voltage clamping the preparation at the MDP during superfusion of 0.2 mM Ba revealed a time-dependent, inwardly directed current. Hyperpolarizing voltage-clamp steps from a holding potential of -50 mV showed that this current was maximal at approximately -70 mV and frequently reversed at membrane potentials of approximately -95 to -115 mV. The time course of this current was biexponential, and the time constant of the faster component decreased with larger hyperpolarization. When the same voltage-clamp protocol was repeated in the presence of 2 mM Ba, no time-dependent current change was detected. In four out of five experiments, Cs (2.5 mM) reduced (but never abolished) the amplitude of the low Ba-induced current. Our results do not support the hypothesis that a hyperpolarization-induced current (iF-like current) is responsible for the automaticity in well-polarized ventricular muscle at low Ba concentrations. Instead, our data suggest that this pacemaker activity is the result of a Ba-induced, time-dependent blockade of the inward rectifier potassium current (iK1).

Automaticity of ventricular fibers induced by low concentrations of barium

The effects of low concentrations of Ba have been studied on dog Purkinje fibers and on guinea pig left ventricular epicardium. In both tissues, Ba induces diastolic depolarization at a membrane potential more negative than -80 mV. This effect becomes clearly detectable at concentrations as low as 10-20 microM in Purkinje fibers bathed in 5.4 mM K Tyrode solution and around 100 microM in ventricular epicardium. In both tissues the Ba-induced depolarization is blocked in a dose-dependent manner by low concentrations of Cs (0.5-5 mM) as is the normal pacemaker current (if) in Purkinje fibers. In ventricular muscle the Ba-induced depolarization develops at the same rate whether the tissue is at rest or depolarized by repetitive stimulation. The maximum (initial) slope of diastolic depolarization is insensitive to D 600, La, and 4-aminopyridine, increased by tetraethylammonium chloride, decreased by tetrodotoxin, and almost entirely suppressed by Na removal. Experimental data and reconstruction of electrical activity suggest that in ventricular muscle the Ba-induced diastolic depolarization results to a large extent from the unmasking of a small pacemaker inward current, mainly carried by Na ions and similar to the pacemaker current of Purkinje fibers. Unmasking of the depolarizing effect of the pacemaker current is very likely due to the decrease by Ba of the inward going rectification. Steady-state inward Na current flowing through the Na window can increase the diastolic depolarization. Residual Cs-insensitive, Ba-induced diastolic depolarization may result from a time-dependent blockade of the background K conductance like the one described in skeletal muscle.

Evidence for a peripheral effect of serotonin or metabolites in 5-hydroxytryptophan-induced hypothermia

The effects of l-5-hydroxytryptophan ( l-5-HTP) on the body temperatures of restrained rats were studied alone and following pretreatment with the central and peripheral decarboxylase inhibitor, benserazide (R04-4602), the peripheral decarboxylase inhibitor, carbidopa (MK-486), or the peripheral serotonergic antagonist, xylamidine tosylate. l-5-Hydroxytryptophan alone (25–200 mg/kg, i.p.) decreased body temperature in a dose-dependent manner. Pretreatment (1 or 4 hr) with benserazide (25–400 mg/kg) produced a dose- and time-dependent blockade of the hypothermic effects of 25 mg/kg and 200 mg/kg of l-5-HTP. Pretreatment with carbidopa (12.5–50 mg/kg) or xylamidine tosylate (1 and 3mg/kg) also produced dose- and time-dependent antagonism of the hypothermic effects of 25 mg/kg of l-5-HTP. Serotonin (5-HT) creatinine sulfate (5–20 mg/kg, i.p.), produced a dose-related hypothermic effect similar to l-5-HTP. Pretreatment with xylamidine tosylate (1 and 3 mg/kg) produced a dose- and time-dependent blockade of the temperature decrease produced by 5 mg/kg of 5-HT. These results suggest that the hypothermic effect produced by systemic l-5-HTP administration in the rat is entirely the result of an effect of serotonin (or metabolites) outside the blood-brain barrier.