Fenvalerate modifies T-type Ca2+ channels in mouse spermatogenic cells

Prior to fertilization sperm must undergo the acrosome reaction that is initiated by opening of T-type Ca(2+) channel. Hence, T-type Ca(2+) channels could be a potential target for agents affecting the acrosome reaction. Our previous data have suggested that fenvalerate, a type II pyrethroid, inhibited the acrosome reaction in mice. To elucidate its potential mechanism we investigated fenvalerate's effect on T-type Ca(2+) channels in mouse pachytene spermatocytes using a whole-cell patch clamp technique. Fenvalerate significantly inhibited T-type Ca(2+) currents in a concentration-dependent manner. The maximal inhibitory concentration was 1 microM. The inhibitory effect of fenvalerate was slow and irreversible after washout of the drug. The curves of activation and inactivation simultaneously shifted to hyperpolarization, about 14 mV, suggesting the open time of Ca(2+) channel was unchanged. Voltage-dependent gating of Ca(2+) channel indicated a change in permeability to ions that contributed to fenvalerate's inhibition on Ca(2+) current. Taken together with our previous findings, these data suggest that the changes of T-type Ca(2+) currents contribute to the suppression of acrosome reaction and fertilizing ability caused by fenvalerate.     

Pathophysiological significance of T-type Ca2+ channels: T-type Ca2+ channels and drug development

T-type Ca(2+) channels are present in cardiovascular, neuronal, and endocrine systems; and they are now receiving attention as novel therapeutic targets. Many drugs and compounds non-specificaly block T-type Ca(2+) channels. Certain dihydropyridine compounds, such as efonidipine, have blocking activity on both L-type and T-type Ca(2+) channels which possibly underlies their excellent clinical profiles such as minimum reflex tachycardia and renal protection. Selective inhibitors of T-type Ca(2+) channels, such as non-hydrolyzable mibefradil and R(-)-efonidipine, are powerful pharmacological tools for further studies and may lead to the development of novel therapeutic strategies.     

Pathophysiological significance of T-type Ca2+ channels: role of T-type Ca2+ channels in renal microcirculation

Since conventional Ca(2+) antagonists, with predominant blockade of L-type voltage-dependent Ca(2+) channels, elicit preferential dilation of afferent arterioles, they might ostensibly aggravate glomerular hypertension. Recently, novel Ca(2+) antagonists, with inhibitory action on L-/T-type Ca(2+) channels, have been reported to dilate both afferent and efferent arterioles. The present review attempted to characterize the renal action of these Ca(2+) antagonists and evaluated the consequences following the treatment with these agents. In contrast to conventional Ca(2+) antagonists (e.g., nifedipine), novel antagonists (e.g., benidipine, efonidipine) potently dilated afferent and efferent arterioles; their action on efferent arterioles appeared to be mediated by the T-type Ca(2+) channel blockade, probably through the inhibition of the intracellular Ca(2+) release. The comparison of the anti-proteinuric action in subtotally nephrectomized rats showed that efonidipine exerted more prominent action than nifedipine. Furthermore, Ca(2+) antagonists with T-type Ca(2+) inhibitory action inhibited renin/aldosterone release and proinflammatory process. Finally, patients with chronic renal disease given a 48-week efonidipine treatment showed reduced proteinuria, and this effect was seen even when mean arterial blood pressure failed to become less than 100 mmHg. Collectively, T-type Ca(2+) channel blockade provides beneficial action in renal injury. Various mechanisms serve to protect against renal injury, including systemic/glomerular hemodynamic action and non-hemodynamic mechanisms.     

Molecular pharmacology of T-type Ca2+ channels

Over the past few years increasing attention has been focused on T-type calcium channels and their possible physiological and pathophysiological roles. Efforts toward elucidating the exact role(s) of these calcium channels have been hampered by the lack of T-type specific antagonists, resulting in the subsequent use of less selective calcium channel antagonists. In addition, the activity of these blockers often varies with cell or tissue type, as well as recording conditions. This review summarizes a variety of compounds that exhibit varying degrees of blocking activity towards T-type Ca2+ channels. It is designed as an aid for researchers in need of antagonists to study the biophysical and pathological nature of T-type channels, as well as a starting point for those attempting to develop potent and selective antagonists of the channel.     

Pathophysiological significance of T-type Ca2+ channels: properties and functional roles of T-type Ca2+ channels in cardiac pacemaking

Calcium channels are essential for excitation-contraction coupling and pacemaker activity in cardiac myocytes. While L-type Ca(2+) channels (LCC) have been extensively studied, functional roles of T-type channels (TCC) in native cardiac myocytes are still debatable. TCC are activated at more negative membrane potentials than LCC and therefore facilitate slow diastolic depolarization in sinoatrial node cells. Recent studies showed that selective inhibition of TCC produced a marked slowing of the pacemaker rhythm, indicating that contribution of TCC to cardiac automaticity was relatively larger than what had been speculated in previous studies. To re-evaluate TCC, we measured current density and kinetics of TCC in sinoatrial node cells of various mammalian species. Current density of TCC was larger in mice and guinea pigs than in rabbit and porcine sinoatrial node cells. Interestingly, few or no obvious TCC were recorded in porcine sinoatrial node cells. Furthermore, it was demonstrated that TCC could be enhanced by several vasoactive substances, thereby increasing spontaneous firing rate of sinoatrial node cells. TCC may, at least in part, account for different heart rates among various mammalian species. In addition, TCC might be involved in physiological and/or pathophysiological modulations of the heart rate.     

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

Aim:

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels and voltage-dependent K⁺ (K_V) channels in rat coronary artery smooth muscle cells (CASMCs).

Methods:

Rat CASMCs were isolated by an enzyme digestion method. BK_Ca and K_V currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK_Ca and K_V channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L.

Results:

When DHA concentrations were greater than 10 μmol/L, BK_Ca currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BK_Ca currents were increased at the above concentrations, respectively. The half-effective concentration (EC₅₀) of DHA on BK_Ca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, K_V currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of K_V currents were blocked at the different concentrations listed above, respectively. The EC₅₀ of DHA on K_V currents was 44.20±0.63 μmol/L.

Conclusion:

DHA can activate BK_Ca channels and block K_V channels in rat CASMCs, and the EC₅₀ of DHA for BK_Ca channels is lower than that for K_V channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK_Ca channels.     

Inhibition of T-type Ca(2+) currents in mouse spermatogenic cells by gossypol, an antifertility compound

Gossypol, a male antifertility compound isolated from cotton, has been proved to inhibit capacitation and the acrosome reaction in human and mammalian sperm. Here, by using whole-cell recording, we observed the effects of gossypol on Ca(2+) and Cl(-) currents in mouse spermatogenic cells obtained by mechanical dissociation. The results showed that gossypol concentration-dependently and irreversibly inhibited T-type Ca(2+) currents in the cells. When the concentration of gossypol was > or =5 microM, the currents were blocked completely. The time to current block was progressively shortened as the gossypol concentration was increased from 5 to 80 microM. Moreover, the drug increased the time constant of inactivation in a concentration-dependent manner, while it did not affect the activation of the current. The inhibitory effect on the T-type Ca(2+) current did not correlate with signaling mediated by G proteins and tyrosine phosphorylation. No obvious effect of gossypol on Cl(-) currents was observed. These data suggest that the gossypol-induced inhibition of T-type Ca(2+) currents could be responsible for the antifertility activity of the compound, indicating a possibility to use gossypol as a local contraceptive drug.     

Acidosis-induced protein tyrosine phosphorylation depends on Ca2+ influx via voltage-dependent Ca2+ channels in SHR aorta

The contractile response to acidosis in isolated aorta from spontaneously hypertensive rat (SHR) depends upon tyrosine phosphorylation of phosphatidylinositol 3 kinase (PI3-kinase) and Ca2+ influx via voltage-dependent Ca2+ channels (VDCC). In this study, verapamil, a VDCC inhibitor, was shown to markedly inhibit acidic pH-induced contraction, whereas the residual contraction in the presence of verapamil was unaffected by the PI3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY-294002). Interestingly, the LY-294002-insensitive component of contraction was further inhibited by verapamil in the presence of LY-294002. Western blotting revealed that acidosis stimulated tyrosine phosphorylation of p85, which was abolished when tissues were pretreated with tyrphostin 23, a tyrosine kinase inhibitor, verapamil or EGTA. In fura-2-loaded aortic strips, acidosis induced a rise in intracellular Ca2+ ([Ca2+]i) that was partially inhibited by LY-294002. The residual increase in [Ca2+]i caused by acidosis in the presence of LY-294002 was abolished by verapamil. These findings suggest that acidosis-induced Ca2+ influx through VDCC is the upstream event leading to the tyrosine phosphorylation of PI3-kinase, which in turn contributes to the enhancement of Ca2+ entry to some extent in SHR aorta.     

Use of transgenic mice to study voltage-dependent Ca2+ channels

During the past decade a great number of genes encoding high- and low-voltage-dependent Ca(2+) channels and their accessory subunits have been cloned. Studies of Ca(2+) channel structure-function relationships and channel regulation using cDNA expression in heterologous expression systems have revealed intricate details of subunit interaction, regulation of channels by protein kinase A (PKA) and protein kinase C (PKC), drug binding sites, mechanisms of drug action, the ion conduction pathway and other aspects of channel function. In recent years, however, we have arrived at the brink of an entirely new strategy to study Ca(2+) channels by overexpressing or knocking out genes encoding these channels in transgenic mice. In this article, various models of gene knockout or gene overexpression will be discussed. This new approach will reveal many secrets regarding Ca(2+) channel regulation and the control of Ca(2+)-dependent cellular processes.     

T-type Ca2+ channels as therapeutic targets in the nervous system

Low-voltage-activated calcium channels, also known as T-type calcium channels, are widely expressed in various types of neurons. In contrast to high-voltage-activated calcium channels which can be activated by a strong depolarization of membrane potential, T-type channels can be activated by a weak depolarization near the resting membrane potential once deinactivated by hyperpolarization, and therefore can regulate the excitability and electroresponsiveness of neurons under physiological conditions near resting states. Recently, the molecular diversity and functional multiplicity of T-type channels have been demonstrated through molecular genetic studies coupled with physiological and behavioral analysis. Understanding the functional consequences of modulation of each subtype of these channels in vivo could point to the right direction for developing therapeutic tools for relevant diseases.     

Effects of demethylzeylasteral and celastrol on spermatogenic cell Ca2+ channels and progesterone-induced sperm acrosome reaction

The male antifertility effect of a water-chloroform extract of Tripterygium wilfordii Hook. f. (GTW) and several monomers isolated from GTW has attracted worldwide interest. In the present study, the effects of two isolated monomers from GTW, demethylzeylasteral and celastrol, on the Ca(2+) channels in mouse spermatogenic cells and on the sperm acrosome reaction were investigated by whole-cell patch-clamp recording and chlortetracycline staining methods, respectively. The results showed that demethylzeylasteral concentration-dependently and in a partially reversible manner inhibited the Ca(2+) current in spermatogenic cells with an IC(50) of 8.8 microg/ml. Celastrol decreased the Ca(2+) current in the cells time-dependently and irreversibly. The changes in the activation and inactivation time constants of Ca(2+) currents after application of these two compounds were also examined. Demethylzeylasteral increased both activation and inactivation time constants of Ca(2+) currents, and celastrol had no significant effect on them. The two compounds also inhibited significantly the sperm acrosome reaction initiated by progesterone. These data suggest that inhibition of Ca(2+) currents could be responsible for the antifertility activity of these compounds.     

Receptor-mediated modulation of voltage-dependent Ca2+ channels via heterotrimeric G-proteins in neurons

The activity of voltage-dependent Ca2+ channels is highly regulated by neurotransmitter receptors coupled to heterotrimeric G-proteins. In the expression studies using cloned Ca2+ channel subunits, it has been clarified that the main mechanism of the inhibition of N-type channel current is mediated directly by G-protein betagamma subunits in a membrane-delimited and voltage-sensitive manner. In addition, recent studies have also clarified that N-type channels are modulated by several G-protein alpha subunits in different ways. Among them, G(alpha o) mediates a voltage-resistant inhibition of N-type current by neurotransmitters. This type of inhibition is more apparent in the case of P/Q-type channels in both native cells and expression systems. Moreover, other G-protein subunits, such as G(alpha q) and G(alpha s), also seem to regulate N-type channels in a membrane-delimited manner. The fine tunings of Ca2+ channel activity by intracellular proteins have physiological and pathological meanings in the regulation of Ca2+ influx into excitable cells by neurotransmitters and pharmacological implications as novel drug targets for controlling Ca2+ influx.     

Targeting Ca2+ channels to treat pain: T-type versus N-type

The transmission of pain signals at the spinal level is crucially dependent on voltage-gated Ca2+ channels in nociceptive neurons. Pharmacological and gene-knockout studies implicate N-type Ca2+ channels as key mediators of nociceptive signaling in dorsal root ganglion (DRG) neurons, and as potential targets for the development of analgesic drugs. Furthermore, nociceptor-specific alternative splicing of the gene encoding N-type Ca2+ channels might provide strategies for splice-isoform-specific drug targeting. More recently, T-type Ca2+ channels have been implicated in the processing of pain signals at both spinal and thalamic levels. However, although inhibition of T-type channel activity in DRG neurons mediates analgesia, gene knockout of T-type channels in the CNS is reported to increase the perception of visceral pain. In this review, we discuss the implications of these findings for the design of novel therapeutic strategies and contrast the role of T-type channels with that of N-type channels in pain transmission and analgesia.     

Antimigraine dotarizine blocks P/Q Ca2+ channels and exocytosis in a voltage-dependent manner in chromaffin cells

The mechanism of blockade of P/Q Ca(2+) channels by antimigraine, dotarizine, was studied in voltage-clamped bovine adrenal chromaffin cells. Inward currents through P/Q channels were pharmacologically isolated by superfusing the cells with omega-conotoxin GVIA (1 microM) plus nifedipine (3 microM). Dotarizine (10-30 microM) blocked the P/Q fraction of I(Ba) and promoted current inactivation. Thus, dotarizine caused a greater blockade of the late I(Ba), compared with blockade of the early peak I(Ba). This effect was more prominent, the longer was the duration of the depolarising pulse. The blockade of I(Ba) was also greater at more depolarising holding potentials (i.e. -60 mV), than was the blockade produced at more hyperpolarising holding potentials (i.e. -80 or -110 mV). Catecholamine secretory responses to brief pulses (2 s) of a Krebs-HEPES solution containing 100 mM K(+) and 2 mM Ca(2+) was blocked by 3 microM dotarizine. Blockade was faster and greater when dotarizine was applied on cells that were previously depolarised with Krebs-HEPES deprived of Ca(2+) and containing increasing concentrations of K(+). This voltage-dependent blockade of P/Q channels and exocytosis might be the underlying mechanism explaining the dotarizine prophylaxis of migraine attacks.     

Contrasting anesthetic sensitivities of T-type Ca2+ channels of reticular thalamic neurons and recombinant Ca(v)3.3 channels

Reticular thalamocortical neurons express a slowly inactivating T-type Ca(2+) current that is quite similar to that recorded from recombinant Ca(v)3.3b (alpha1Ib) channels. These neurons also express abundant Ca(v)3.3 mRNA, suggesting that it underlies the native current. Here, we test this hypothesis by comparing the anesthetic sensitivities of recombinant Ca(v)3.3b channels stably expressed in HEK 293 cells to native T channels in reticular thalamic neurons (nRT) from brain slices of young rats. Barbiturates completely blocked both Ca(v)3.3 and nRT currents, with pentobarbital being about twice more potent in blocking Ca(v)3.3 currents. Isoflurane had about the same potency in blocking Ca(v)3.3 and nRT currents, but enflurane, etomidate, propofol, and ethanol exhibited 2-4 fold higher potency in blocking nRT vs Ca(v)3.3 currents. Nitrous oxide (N(2)O; laughing gas) blocked completely nRT currents with IC(50) of 20%, but did not significantly affect Ca(v)3.3 currents at four-fold higher concentrations. In addition, we observed that in lower concentration, N(2)O reversibly increased nRT but not Ca(v)3.3 currents. In conclusion, contrasting anesthetic sensitivities of Ca(v)3.3 and nRT T-type Ca(2+) channels strongly suggest that different molecular structures of Ca(2+) channels give rise to slowly inactivating T-type Ca(2+) currents. Furthermore, effects of volatile anesthetics and ethanol on slowly inactivating T-type Ca(2+) channel variants may contribute to the clinical effects of these agents.     

Contrasting the effects of nifedipine on subtypes of endogenous and recombinant T-type Ca2+ channels

There is evidence that nifedipine (Nif) - a dihydropyridine (DHP) Ca(2+)-channel antagonist mostly known for its L-type-specific action--is capable of blocking low voltage-activated (LVA or T-type) Ca(2+) channels as well. However, the discrimination by Nif of either various endogenous T-channel subtypes, evident from functional studies, or cloned Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 T-channel alpha 1 subunits have not been determined. Here, we investigated the effects of Nif on currents induced by Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 expression in Xenopus oocytes or HEK-293 cells (I(alpha 1G), I(alpha 1H) and I(alpha 1I), respectively) and two kinetically distinct, "fast" and "slow", LVA currents in thalamic neurons (I(LVA,f) and I(LVA,s)). At voltages of the maximums of respective currents the drug most potently blocked I(alpha 1H) (IC(50)=5 microM, max block 41%) followed by I(alpha 1G) (IC(50)=109 microM, 23%) and I(alpha 1I) (IC(50)=243 microM, 47%). The mechanism of blockade included interaction with Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 open and inactivated states. Nif blocked thalamic I(LVA,f) and I(LVA,s) with nearly equal potency (IC(50)=22 microM and 28 microM, respectively), but with different maximal inhibition (81% and 51%, respectively). We conclude that Ca(v)3.2 is the most sensitive to Nif, and that quantitative characteristics of drug action on T-type Ca(2+) channels depend on cellular system they are expressed in. Some common features in the voltage- and state-dependence of Nif action on endogenous and recombinant currents together with previous data on T-channel alpha 1 subunits mRNA expression patterns in the thalamus point to Ca(v)3.1 and Ca(v)3.3 as the major contributors to thalamic I(LVA,f) and I(LVA,s), respectively.     

5beta-reduced neuroactive steroids are novel voltage-dependent blockers of T-type Ca2+ channels in rat sensory neurons in vitro and potent peripheral analgesics in vivo

T-type Ca(2+) channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5alpha-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca(2+) channels and cause analgesia in a thermal nociceptive model (Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5beta-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca(2+) channel function in vitro. We found that most of the steroids completely blocked T-type Ca(2+) currents in vitro with IC(50) values at a holding potential of -90 mV ranging from 2.8 to 40 microM. T current blockade exhibited mild voltage-dependence, suggesting that 5beta-reduced neuroactive steroids stabilize inactive states of the channel. For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED(50), 30 ng/100 microl) obtained with a compound [(3beta,5beta,17beta)-3-hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5alpha-reduced steroids, these 5beta-reduced steroids are more efficacious blockers of neuronal T-type Ca(2+) channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca(2+) channels in peripheral pain pathways.     

Tributyltin-induced Ca(2+) mobilization via L-type voltage-dependent Ca(2+) channels in PC12 cells

The effects of tributyltin (TBT) on cytosolic Ca(2+) concentration ([Ca(2+)](c)) and cell viability were investigated in nerve growth factor-differentiated PC12 cells. TBT concentration dependently increased [Ca(2+)](c) with an EC(50) value of 0.07μM. This effect was markedly reduced by removal of the extracellular Ca(2+) or membrane depolarization with a high K(+) medium, but unaffected by thapsigargin causing depletion of intracellular Ca(2+) stores. The L-type voltage-dependent Ca(2+) channel (VDCC) blocker nicardipine blocked the effect of TBT, but the N-type VDCC blocker ω-conotoxin did not. TBT decreased the number of viable cells with an EC(50) value of 0.09μM. The TBT-induced cell death was prevented by nicardipine or by chelating the cytosolic Ca(2+) with BAPTA-AM, but not by ω-conotoxin. The results show that TBT causes an increase in [Ca(2+)](c) via activating L-type VDCCs, and support the idea that the organotin-induced cell death arises through Ca(2+) mobilization via L-type VDCCs.