Specific Ion Channel Blockers Research Articles

A dynamic clamping approach using in silico IK1 current for discrimination of chamber-specific hiPSC-derived cardiomyocytes

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents.

Channelopathies in Breast Cancer and Gynecological Cancers

Increasing evidence shows that ion channels play a significant role in cell proliferation, migration, apoptosis and differentiation. Many research works in gynecological cancer suggest that ion channels are involved in aberrant tumor growth and upregulation or downregulation of ion channels results in tumor growth arrest. Channelopathies are a diverse set of inherited mutations of ion channels that result in altered biophysical properties. In normal cells, there is a switch between cell growth and cell death which apparently depend upon the temporal organization and magnitude of different ion channels. There is a need for specific ion channel blockers which can abrogate the cellular mechanism of the cancer cell. Typically the impact of ion channels on cancer depends upon the magnitude and temporal organization of the ion channel activation and the activity of other signaling mechanisms. Membrane proteins are responsible for ionic homeostasis in ion channels. As cancer can be linked with the altered biophysical properties of ion channels a border context of different ion channels in cancer seems absolutely appropriate. The field of onco-channelopathies is rapidly expanding and understanding the pathophysiological mechanisms underlying the development of cancer enables researchers to better diagnose and develop treatment options for cancer. This review focuses on the role of ion channels in breast cancer and other gynecological cancers including ovarian cancer and cervical cancer, and how they contribute to tumor development.

Transgenic LQT2, LQT5, and LQT2-5 rabbit models with decreased repolarisation reserve for prediction of drug-induced ventricular arrhythmias.

Background and PurposeReliable prediction of pro‐arrhythmic side effects of novel drug candidates is still a major challenge. Although drug‐induced pro‐arrhythmia occurs primarily in patients with pre‐existing repolarisation disturbances, healthy animals are employed for pro‐arrhythmia testing. To improve current safety screening, transgenic long QT (LQTS) rabbit models with impaired repolarisation reserve were generated by overexpressing loss‐of‐function mutations of human HERG (HERG‐G628S, loss of IKr; LQT2), KCNE1 (KCNE1‐G52R, decreased IKs; LQT5), or both transgenes (LQT2‐5) in the heart.Experimental ApproachEffects of K+ channel blockers on cardiac repolarisation and arrhythmia susceptibility were assessed in healthy wild‐type (WT) and LQTS rabbits using in vivo ECG and ex vivo monophasic action potential and ECG recordings in Langendorff‐perfused hearts.Key ResultsLQTS models reflect patients with clinically “silent” (LQT5) or “manifest” (LQT2 and LQT2‐5) impairment in cardiac repolarisation reserve: they were more sensitive in detecting IKr‐blocking (LQT5) or IK1/IKs‐blocking (LQT2 and LQT2‐5) properties of drugs compared to healthy WT animals. Impaired QT‐shortening capacity at fast heart rates was observed due to disturbed IKs function in LQT5 and LQT2‐5. Importantly, LQTS models exhibited higher incidence, longer duration, and more malignant types of ex vivo arrhythmias than WT.Conclusion and ImplicationsLQTS models represent patients with reduced repolarisation reserve due to different pathomechanisms. As they demonstrate increased sensitivity to different specific ion channel blockers (IKr blockade in LQT5 and IK1 and IKs blockade in LQT2 and LQT2‐5), their combined use could provide more reliable and more thorough prediction of (multichannel‐based) pro‐arrhythmic potential of novel drug candidates.

P2559The mechanisms of spontaneous action potential generation in sinoauricular valve of the chick embryos

Abstract Background The changes in the density of ion channels and/or damage of ion channel proteins violates the homeostasis of the potential forming ions in myocardium cells and may contribute to arrhythmogenesis. The study of ion channel function in embryos is important in order to understand the mechanisms of arrhythmia. The purpose of the present study was to identify the key ion channels that form action potentials (APs) in pacemaker cells in the area of the sinoauricular (SA) chicken embryo valve. Material and methods The embryos were removed from fertilized White Leghorn breed chicken eggs on the 10±1 day of incubation (ED10). The experiments were performed on freshly prepared strips (3 mm × 2 mm) of the right atrium, which included the left SA valve itself as well as the surrounding tissue. After that the APs were recorded from the area of left SA valve using the microelectrode technique. The functional analysis of ion channels was performed using the specific ion channel blockers. Results The strips generated APs with slow diastolic depolarization (SDD) and frequency from 132 to 251 impulses per min (ipm) (average - 177±22 ipm). The APs amplitude and dV/dtmax were 102±9 mV and 98±25 V/s (nstrips=49). At the third minute of exposure to E-4031 (1 μmol), a blocker of rapid outward K-current channels, IKr, the increase in the duration of the AP repolarization phase by 30% and the shortening of SDD by 20–25% (p<0.05; n=6) were registered. The APs generation frequency increased by 10%. Subsequently we observed the monotonic decrease in the AP amplitude due to the reduced overshoot and depolarized Emax. The electrical activity of all strips was blocked on the 20±2 min (Fig.1.a-c). Lidocaine (500 μmol), a blocker of Nav1.5 channels, increased a duration of SDD and APD90 twice, but decreased dV/dtmax by 2.5 times. The APs generation frequency decreased by 30% in comparison to the control. After 8±2 min exposure to lidocaine, the APs generation was blocked in 56% of samples. It is important to mention that in these strips right before the APs generation was blocked dV/dtmax was 30±15 V/s. In the samples, where the APs were not blocked after the exposure to lidocaine this parameter was 14±10 V/s (Fig. 1d-e). Nifedipine (10 μmol), a blocker of L-type Ca2+-channels, decreased APD90 by 56% and SDD by 25%. The APs generation frequency increased twice in comparison to the control samples (fig.1f-h). The chronotropic effects of nifedipine were dose-dependent. Nifedipine did not induce the blocking of APs generation. Figure 1. Effects of E-4031 (a-c), lidocaine (d-e) and nifedipine (e-g) on APs. Conclusions The received data demonstrate that the key role in the APs formation in ED10 embryos belong to the outward IKr and inward INa, but not to ICa,L. Thus, the mechanisms of spontaneous action potential generation in ED10 chick embryos have more in common with that in human iPSC-derived cardiomyocytes rather than in SA area cells of the ED11 mice embryos. Acknowledgement/Funding Rus fund Basic Research No. 18-34-00654; Project RAS No. 0415-2019-0006 and No. AAAA-A17-117012310152-2

Methylglyoxal causes pain and hyperalgesia in human through C-fiber activation.

The endogenous metabolite methylglyoxal (MG) accumulates in diabetic patients with neuropathic pain. Methylglyoxal could be a mediator of diabetes-induced neuropathic pain through TRPA1 activation and sensitization of the voltage-gated sodium channel subtype 1.8. In this study, we tested the algogenic and sensitizing effect of MG in healthy human subjects using intracutaneous microinjections. The involvement of C fibers was assessed through selective A-fiber nerve block, axon-reflex-erythema, and through single nerve fiber recordings in humans (microneurography). Involvement of the transduction channels TRPA1 and TRPV1 in MG-induced pain sensation was investigated with specific ion channel blockers. We showed for the first time in healthy humans that MG induces pain, axon-reflex-erythema, and long-lasting hyperalgesia through the activation of C nociceptors. Predominantly, the subclass of mechano-insensitive C fibers is activated by MG. A fibers contribute only negligibly to the burning pain sensation. Selective pharmacological blockade of TRPA1 or TRPV1 showed that TRPA1 is crucially involved in MG-induced chemical pain sensation and heat hyperalgesia. In conclusion, the actions of MG through TRPA1 activation on predominantly mechano-insensitive C fibers might be involved in spontaneously perceived pain in diabetic neuropathy and hyperalgesia as well as allodynia.

Origin and classification of spontaneous discharges in mouse superficial dorsal horn neurons

Superficial laminae of the spinal cord possess a considerable number of neurons with spontaneous activity as reported in vivo and in vitro preparations of several species. Such neurons may play a role in the development of the nociceptive system and/or in the spinal coding of somatosensory signals. We have used electrophysiological techniques in a horizontal spinal cord slice preparation from adult mice to investigate how this activity is generated and what are the main patterns of activity that can be found. The results show the existence of neurons that fire regularly and irregularly. Within each of these main types, it was possible to distinguish patterns of spontaneous activity formed by single action potentials and different types of bursts according to intra-burst firing frequency. Activity in neurons with irregular patterns was blocked by a mixture of antagonists of the main neurotransmitter receptors present in the cord. Approximately 82% of neurons with a regular firing pattern were insensitive to synaptic antagonists but their activity was inhibited by specific ion channel blockers. It is suggested that these neurons generate endogenous activity due to the functional expression of hyperpolarisation-activated and persistent sodium currents driving the activity of irregular neurons.

Ion channel blockers and glioblastoma risk and outcome: a nested case-control and retrospective cohort studies.

Mutations in ion channels are common among patients with glioblastoma multiforme (GBM) and promote cell migration and invasion. We sought to evaluate the association between the use of specific ion channel blockers such as digoxin, amiodarone, diltiazem and verapamil and GBM risk and survival. We conducted a nested case-control study in a large primary care database from the UK. Cases were defined as all individuals with incident diagnosis of GBM during follow-up. For each case, up to four controls were selected using incidence density sampling. The primary exposure of interest was active treatment with each of the four ion channel blockers. We used conditional logistic regression to estimate odds ratios and 95% confidence interval (CI) for the association between ion channel blocker use and GBM risk. We then performed a Cox regression analysis among those diagnosed with GBM in order to evaluate the association between use of ion channel blockers and overall survival. Both analyses were adjusted to common confounders. The study included 1076 cases and 4253 matched controls. There was no statistically significant difference between cases and controls in cardiac and metabolic risk factors. There was no change in GBM risk in active users of ion channel blockers compared with non-users. Among patients with GBM, active users of amiodarone had worse survival compared with never users with an HR of 4.41 (95%CI 1.95-9.96). There was no statistically significant change in survival among diltiazem, verapamil or digoxin users. Treatment with specific ion channel blockers was not associated with the risk of GBM but was associated with worse survival in patients with GBM. Copyright © 2016 John Wiley & Sons, Ltd.

The Anti-Proliferative Effect of Cation Channel Blockers on T Lymphocytes Stimulated by Anti-CD3 and Anti-CD28

The ion channels of T lymphocytes form a crucial part of the healthy immune system, as they are important for cellular activation and proliferation. In autoimmune diseases, effector memory T cells have a unique ion channel pattern, therefore they are promising therapeutic targets. A number of ion channel inhibitors are known as selective inhibitors of T lymphocyte proliferation, but the data available is contradictory. Our aim was to elucidate this phenomenon by investigating how the blockage of ion channels affects the activation and proliferation of T cells treated previously with different concentrations of mitogens.In our experiments human peripheral blood lymphocytes from volunteers were activated via monoclonal antibodies affecting the TCR-CD3 complex on the cell surface and the co-stimulator molecule CD28. We applied specific ion channel blockers acting on the major cationic channels of the T cell, the Kv1.3, the KCa1.1 and the CRAC channel, either alone or in combination with rapamycin, the inhibitor of the mammalian target of rapamycin (mTOR). Five days after the stimulus flow cytometry measurements were performed to determine the extent of cellular viability and proliferation.Our measurements indicated that ion channel blockers and rapamycin had a negative dose-dependent effect on the amount of cell division. Simultaneous application of blockers for each channel along with rapamycin proved to be the most effective, which indicates that they affect independent regulation pathways. Upon increasing the rate of stimulation, the anti-proliferative effect of the blockers diminished. This phenomenon was unknown to date and may prove to be important in understanding the fine-tuning of T cell activation.

Pharmacological actions of the slow release hydrogen sulfide donor GYY4137 on phenylephrine-induced tone in isolated bovine ciliary artery

Hydrogen sulfide (H2S), a colorless gas characterized by its pungent odor of rotten eggs has been reported to elicit relaxation effects on basal and pre-contracted non-ocular smooth muscles of several mammalian species. In the present study, we investigated the pharmacological actions of a H2S donor, GYY4137 on isolated bovine posterior ciliary artery after contraction with the adrenergic receptor agonist, phenylephrine. Furthermore, we studied the underlying mechanism of inhibitory action of GYY4137 on the posterior ciliary arteries. Isolated bovine posterior ciliary arteries were mounted in oxygenated organ baths and changes in isometric tension were measured with a Grass FT03 transducer connected to a recorder using a Grass Polyview Software. The relaxant actions of GYY4137 on phenylephrine pre-contracted arteries were observed in the absence and presence of an inhibitor of cyclo-oxygenase, flurbiprofen. Furthermore, the inhibitory effects of GYY4137 were studied in the absence or presence of inhibitors/activators of biosynthetic enzymes for H2S and nitric oxide production, as well as specific ion channel blockers. In the concentration range, 100 nM to 100 μM, GYY4137 elicited a concentration-dependant relaxation of phenylephrine-induced tone in isolated posterior ciliary arteries, with IC50 value of 13.4 ± 1.9 μM (n = 6). The cyclo-oxygenase inhibitor, flurbiprofen, significantly (p < 0.01) enhanced the relaxation induced by GYY4137 yielding IC50 value of 0.13 ± 0.08 μM (n = 6). Both the inhibitors of cystathionine β-synthase (aminooxyacetic acid, AOAA, 30 μM) and cystathionine γ-lyase (propargylglycine, PAG, 1 mM) caused significant (p < 0.05) rightward shifts in the concentration–response curve to GYY4137. Furthermore, the KATP channel antagonist, glibenclamide (100 μM) significantly (p < 0.01) attenuated the relaxant action induced by GYY4137 on bovine ciliary artery. Conversely, the activator of cystathionine β-synthase, SAM (100 μM) and an inhibitor of nitric oxide synthase, l-NAME (100 μM) had no significant effect on relaxations induced by GYY4137. We conclude that the inhibitory action of GYY4137 on isolated bovine ciliary artery is dependent upon the endogenous production of both prostanoids and H2S. Furthermore, the observed vascular smooth muscle relaxation induced by GYY4137 is mediated, at least in part, by KATP channels.

Comparative effects of sodium channel blockers in short term rat whole embryo culture

This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the l-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the l-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3h.The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB>bupivacaine>AZA>lidocaine>nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage.The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart.

Antihypertensive Antagonists of L-Type Calcium Channel Exert Enhancement of Alzheimer's Aβ Peptides Effect on Cells

The Alzheimer's Aβ peptides are shown to increase the cytosolic calcium concentrations of cells in culture. Such an alteration may cause a variety of secondary effects, leading to cell degeneration and reduced cell culture growth. Cytosolic calcium concentration can be increased by the formation of cation-selective ion channels by the Aβ peptides. This property is well documented and has been extensively studied both in artificial membrane systems as well as in living cells. Here we show that L-type calcium channel antagonists, commonly used in the treatment of hypertension and ischemic heart disease, intensify the capacity of Aβ peptides to increase cytosolic calcium concentrations and consequently enhance the cytotoxicity of the Aβ peptides. Calcium imaging experiments were conducted with the fluorescent indicator Fura-2 to measure at different periods of times the levels of cytosolic calcium during exposure to Aβ peptides and to the L-type calcium channel antagonists. Two different 1,4-dihydropyridines (nifedipine, and nitrendipine), and Diltiazem, a structurally unrelated Ca2+-channel antagonist belonging to the phenylalkylamine class were used and found to exert significant excitatory effects on the toxicity of Aβ. The reduced toxicity observed in solutions of combinations of Aβ40 and Aβ42 is also reversed by L-type calcium channel antagonists. The toxicity of Aβ peptides was weighed by quantifying the growth of cultures of PC12 cells using the colorimetric assay XTT and by measuring the release of LDH to the media. The enhancement of the Aβ peptides toxicity by L-type calcium channel antagonists can be constrained by specific Aβ ion channel blockers suggesting the mediation of the ion channel property of Aβ peptides. The relevance of our finding derives from the widespread clinical use of L-type calcium channel antagonists in the treatment of hypertension and angina pectoris.

Synchronization through nonreciprocal connections in a hybrid hippocampus microcircuit

Synchronization among neurons is thought to arise from the interplay between excitation and inhibition; however, the connectivity rules that contribute to synchronization are still unknown. We studied these issues in hippocampal CA1 microcircuits using paired patch clamp recordings and real time computing. By virtually connecting a model interneuron with two pyramidal cells (PCs), we were able to test the importance of connectivity in synchronizing pyramidal cell activity. Our results show that a circuit with a nonreciprocal connection between pyramidal cells and no feedback from PCs to the virtual interneuron produced the greatest level of synchronization and mutual information between PC spiking activity. Moreover, we investigated the role of intrinsic membrane properties contributing to synchronization where the application of a specific ion channel blocker, ZD7288 dramatically impaired PC synchronization. Additionally, background synaptic activity, in particular arising from NMDA receptors, has a large impact on the synchrony observed in the aforementioned circuit. Our results give new insights to the basic connection paradigms of microcircuits that lead to coordination and the formation of assemblies.

Immunopharmacology: utilizing antibodies as ion channel modulators

Development of the patch clamp technique by the Nobel Prize winners Bert Sakmann and Erwin Neher led to huge advances in ion channel research. Their work laid the foundations and revolutionized electrophysiological studies of cells and ion channels. These ion channels underlie many basic cellular physiological processes and, therefore, are key therapeutic targets for pharmaceutical companies. However, current pharmacological strategies are hampered by the lack of specific ion channel blockers. Intense research and development programs are now actively employing antibodies to target ion channels in various formats. This review discusses the use of ion channel antibodies and their associated small molecules as pharmacological tools, termed immunopharmacology. In addition, we will review some recent studies looking into clinical applications of immunopharmacology and intrabodies.

Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca2+ cycling and IKACh

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a G(i)-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current (I(f)) or L-type Ca(2+) currents (I(Ca,L)), respectively; and 2) direct G(i)-coupled activation of ACh-activated potassium current (I(KACh)). More recent studies, however, have indicated that Ca(2+) cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca(2+) clock) generates rhythmic, spontaneous local Ca(2+) releases (LCR) that are AC-PKA dependent. LCRs activate Na(+)-Ca(2+) exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was approximately 10 nM, half maximal inhibition (IC(50)) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. G(i) inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that I(f) blockade did not affect BRR at any [CCh] and that I(KACh) activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At IC(50), CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by approximately 50%. The dose response of BRR to CCh in the presence of I(KACh) blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At IC(50), CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of IC(50) CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca(2+) cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulation-induced BRR is entirely dependent on G(i) activation and the extent of G(i) coupling to Ca(2+) cycling via PKA signaling or to I(KACh): at low [CCh], I(KACh) activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca(2+) signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca(2+) signaling and I(KACh) activation underlies a more pronounced BRR.

Identifying needs and opportunities for advancing translational research in cardiovascular disease

In Western Europe, as in the USA, mortality of cardiovascular disease (CVD) has declined significantly in the last 30 years. Life expectancy for patients with coronary heart disease in the USA increased on average by 3 years between 1970 and 2000.1 Several epidemiological analyses support this positive evolution. An analysis of the underlying causes identifies improvements in quality of care and treatment as a major cause, accounting from 50 up to 75% of the success depending on the study samples, the remainder being accounted for by changes in lifestyle and prevention.2 Research and development in several areas of CVD have contributed to this success. In acute coronary events, the identification and development of efficient and safe thrombolytic agents followed by percutaneous coronary intervention with stent implantation were milestones in the reduction of acute mortality and salvage of myocardium.3–5 Statins have brought a major advance in preventing the progression of atherosclerotic disease and have been recognized for their wide mode of action.6 In heart failure (HF), beta-blockers and ACE-inhibitors have increased life expectancy by a leap.7 Some of these improvements have been the result of a classic bench-to-bedside development of a targeted treatment, such as the statins, others have known a more serendipitous development, such as beta-blockers. In other areas, progress in terms of treatment has been less spectacular and fraught with difficulties. Development of antiarrhythmic agents received a large boost from basic insights into cardiac ion channel structure and function, but translation into pharmacology unveiled unexpected pro-arrhythmia risks of some of the highly specific ion channel blockers in class III.8 Automatic defibrillators have taken an important role in the treatment of life-threatening arrhythmias and save lives, but are not without burden and come at a high cost.9 This latter example is …

Mechanosensitive N -Methyl- d -Aspartate Receptors Contribute to Sensory Activation in the Rat Renal Pelvis

The N-methyl-D-aspartate (NMDA) subtype of the ionotropic glutamate receptor is found in the periphery. The present study tested whether NMDA receptors (NMDARs) are present in the ends of afferent renal nerves in the renal pelvis, an area concerned mainly with transmitting sensation and the to reflex regulation of body fluid. The main NMDAR subunit, NMDAzeta1, was found to be more abundant in the renal pelvis than the renal cortex and medulla, and was mainly colocalized with the pan-neuronal marker PGP9.5 or the sensory nerve marker, the neurokinin-1 receptor. However, NMDAzeta1 mRNA was undetectable, suggesting that it might be synthesized outside the renal pelvis. Intrarenal arterial administration of the specific ion channel blocker (+)-MK-801, but not the inactive enantiomer (-)-MK-801, decreased urine output and sodium excretion. High doses of (+)-MK-801 also caused regional vasoconstriction in the renal cortex, as determined by laser-Doppler flowmetry. Intrapelvic administration of the NMDAR ligand D-serine caused a dose-dependent increase in substance P (SP) release and afferent renal nerve activity, but had no effect on arterial pressure. The D-serine-induced sensory activation and SP release were abrogated by (+)-MK-801, the SP receptor blocker L-703,606, or dorsal rhizotomy. Increasing intrapelvic pressure resulted in an increase in afferent renal nerve activity and a diuretic/natriuretic response. Interestingly, these effects were attenuated by prior administration of (+)-MK-801. These results indicate that NMDAR-positive sensory nerves are present in the renal pelvis and contribute to the renorenal reflex control of body fluid.

Plasma membrane ion channels in suicidal cell death

The machinery leading to apoptosis includes altered activity of ion channels. The channels contribute to apoptotic cell shrinkage and modify intracellular ion composition. Cl − channels allow the exit of Cl −, osmolytes and HCO 3 - leading to cell shrinkage and cytosolic acidification. K + exit through K + channels contributes to cell shrinkage and decreases intracellular K + concentration, which in turn favours apoptotic cell death. K + channel activity further determines the cell membrane potential, a driving force for Ca 2+ entry through Ca 2+ channels. Ca 2+ may enter through unselective cation channels. An increase of cytosolic Ca 2+ may stimulate several enzymes executing apoptosis. Specific ion channel blockers may either promote or counteract suicidal cell death. The present brief review addresses the role of ion channels in the regulation of suicidal cell death with special emphasis on the role of channels in CD95 induced apoptosis of lymphocytes and suicidal death of erythrocytes or eryptosis.

Modulation of neurotrophic growth factors as a therapeutic strategy for neuropathic pain

AbstractNeurotrophic growth factors have tremendous untapped potential for the treatment of neurological injuries and diseases. In contrast to specific neurotransmitter modulators or ion channel blockers, a neurotrophic growth factor profoundly impacts multiple molecular pathways within a cell, with the possibility of exerting broad, and potentially “disease‐modifying” effects that may counteract the underlying pathology. Although numerous clinical trials have been carried out with the prototypic neurotrophic growth factors, including most notably NGF for diabetic neuropathy and GDNF for Parkinson's disease, none of these trials have been successful to date in providing compelling data for drug approval. The apparent lack of translation from the bench to the clinic may be considered in the context of our current understanding of the neurotrophic growth factors, especially mechanism of action, efficacy in animal models of disease, and safety and tolerability, and how these factors relate to clinical trial design. The application of this new knowledge to future clinical trials of neuropathic pain may provide opportunities for successful clinical development of neurotrophic growth factor modulators. Drug Dev. Res. 67:389–403, 2006. © 2006 Wiley‐Liss, Inc.

Peptide‐Specific Antibody as a Tool to Evaluate TRPM3 Ion Channel Function

Calcium channel blockers can be used to selectively inhibit calcium influx across a membrane, making them potentially useful as therapeutic tools. Existing transient receptor potential (TRP) channel blockers are relatively non-specific, but have previously been used to characterise individual members of the TRP family. The activation of TRP channels can be more specifically inhibited through the use of anti-peptide antibodies targeted to particular regions of the channel structure (Xu & Beech, 2001. Circ. Res. 88:84–87). The polyclonal antibody TM3E3 was designed to specifically bind to a peptide corresponding to the 3rd extracellular loop of TRPM3, a member of the melastatin-like TRP family. The location and sequence of the peptide were determined using hydrophilicity analysis of the channel sequence (Xu et al., 2005. Nat. Biotechnol. 23:1289–1293). Here we use Ca2+-imaging to study the specificity of TM3E3 for human TRPM3 expressed in HEK-293 cells. Extracellular application of the antibody has a functional effect, inhibiting the sphingosine-induced activation of TRPM3. It does not inhibit activation of the closely related channel TRPM2 or the canonical family member TRPC5. TM3E3 will serve as a useful tool for further characterisation of endogenous TRPM3 in a variety of cells and tissues, and more importantly the results strengthen the idea that E3 targeting may be used to generate specific ion channel blockers for a variety of channels, with therapeutic potential. This work is supported by the BBSRC, the Welcome Trust and the BHF. Many thanks to C. Harteneck for providing the TRPM3 clone.

Physiological volume regulation by spermatozoa

Maturing spermatozoa passing through the epididymis experience increasing osmolality in the luminal environment and mature cells are stored in fluids hyper-osmotic to serum. When ejaculated into the female tract, they encounter a hypo-osmotic challenge which initiates the process of regulatory volume decrease (RVD). Defects in RVD result in hindrance of mucus penetration in man and failure of utero-tubal passage in mice. Epididymal sperm from the mouse and cynomolgus monkey and ejaculated sperm from man and monkey have been isolated and dispersed in media with osmolalities mimicking those of uterine fluid or cervical mucus. The effects of specific and broad-spectrum ion channel blockers indicate the involvement of separate K + and Cl − channels as well as organic osmolytes in physiological sperm RVD, with mechanisms developed during epididymal maturation. Western blotting and immuno-cytochemistry identify and localise some of these channels which play a crucial role in fertilisation in vivo and could be targets for post-testicular contraception.