Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Molecular requirements for L-type Ca2+ channel blockade by testosterone

Despite being generally perceived as detrimental to the cardiovascular system, testosterone has marked beneficial vascular effects; most notably it acutely and directly causes vasodilatation. Indeed, men with hypotestosteronaemia can present with myocardial ischemia and angina which can be rapidly alleviated by infusion of testosterone. To date, however, in vitro studies have failed to provide a convincing mechanism to account for this clinically important effect. Here, using whole-cell patch-clamp recordings to measure current flow through recombinant human L-type Ca2+ channel alpha(1C) subunits (Ca(v)1.2), we demonstrate that testosterone inhibits such currents in a concentration-dependent manner. Importantly, this occurs over the physiological range of testosterone concentrations (IC50 34 nM), and is not mimicked by the metabolite 5alpha-androstan-17beta-ol-3-one (DHT), nor by progesterone or estradiol, even at high (10 microM) concentration. L-type Ca2+ channels in the vasculature are also important clinical targets for vasodilatory dihydropyridines. A single point mutation (T1007Y) almost completely abolishes nifedipine sensitivity in our recombinant expression system. Crucially, the same mutation renders the channels insensitive to testosterone. Our data strongly suggest, for the first time, the molecular requirements for testosterone binding to L-type Ca2+ channels, thereby supporting its beneficial role as an endogenous Ca2+ channel antagonist in the treatment of cardiovascular disease.

Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound

In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.

Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Antisense oligonucleotides against α1E reduce R-type calcium currents in cerebellar granule cells

Many neurons of the central nervous system display multiple high voltage-activated Ca2+ currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the α1E subunit, which yields Ca2+ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against α1E could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6–8 days in vitro. Incubation with α1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from −10 ± 0.6 picoamperes/picofarad (pA/pF) (n = 6) in the untreated controls to −4.8 ± 0.8 pA/pF (n = 11) (P < 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (−11.3 ± 3.2 pA/pF). The specificity of the α1E antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against α1A did not affect R-type current expression (−11.5 ± 1.7 and −11.7 ± 1.7 pA/pF, respectively), whereas the α1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of α1 subunits support the high voltage-activated R-type current in cerebellar granule cells.

Predominance of the α1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken’s cochlea

The voltage-gated Ca2+ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca2+ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca2+ channels, we investigated the expression of L-type α1 subunits in hair cells of the chicken’s cochlea. In PCRs with five different pairs of degenerate primers, we always obtained α1D products, but only once an α1C product and never an α1S product. A full-length α1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the α1D subunit was about 90% identical to those of the mammalian α1D subunits. In situ hybridization confirmed that the α1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the α1D mRNA is 100–500 times more abundant than the α1C mRNA. We conclude that most, if not all, voltage-gated Ca2+ channels in hair cells contain an α1D subunit. Furthermore, we propose that the α1D subunit plays a hitherto undocumented role at tonic synapses.

Multiple calcium channel transcripts in rat osteosarcoma cells: selective activation of alpha 1D isoform by parathyroid hormone.

Osteoblasts express calcium channels that are thought to be involved in the transduction of extracellular signals regulating bone metabolism. The molecular identity of the pore-forming subunit (alpha 1) of L-type calcium channel(s) was determined in rat osteosarcoma UMR-106 cells, which express an osteoblast phenotype. A homology-based reverse transcriptase-polymerase chain reaction cloning strategy was employed that used primers spanning the fourth domain. Three types of cDNAs were isolated, corresponding to the alpha 1S (skeletal), alpha 1C (cardiac), and alpha 1D (neuroendocrine) isoforms. In the transmembrane segment IVS3 and the extracellular loop formed by the IVS3-S4 linker, a single pattern of mRNA splicing was found that occurs in all three types of calcium channel transcripts. Northern blot analysis revealed an 8.6-kb mRNA that hybridized to the alpha 1C probe and 4.8- and 11.7-kb mRNAs that hybridized to the alpha 1S and alpha 1D probes. Antisense oligonucleotides directed to the calcium channel alpha 1D transcript, but not those directed to alpha 1S or alpha 1C transcripts, inhibited the rise of intracellular calcium induced by parathyroid hormone. However, alpha 1D antisense oligonucleotides had no effect on the accumulation of cAMP induced by parathyroid hormone. When L-type calcium channels were activated with Bay K 8644, antisense oligonucleotides to each of the three isoforms partially inhibited the rise of intracellular calcium. The present results provide evidence for the expression of three distinct calcium channel alpha 1-subunit isoforms in an osteoblast-like cell line. We conclude that the alpha 1D isoform is selectively activated by parathyroid hormone.

Differential involvement of N-type calcium channels in transmitter release from vasoconstrictor and vasodilator neurons

1 The effects of calcium channel blockers on co-transmission from different populations of autonomic vasomotor neurons were studied on isolated segments of uterine artery and vena cava from guinea-pigs. 2 Sympathetic, noradrenergic contractions of the uterine artery (produced by 200 pulses at 1 or 10 Hz; 600 pulses at 20 Hz) were abolished by the N-type calcium channel blocker omega-conotoxin (CTX) GVIA at 1-10 nM. 3 Biphasic sympathetic contractions of the vena cava (600 pulses at 20 Hz) mediated by noradrenaline and neuropeptide Y were abolished by 10 nM CTX GVIA. 4 Neurogenic relaxations of the uterine artery (200 pulses at 10 Hz) mediated by neuronal nitric oxide and neuropeptides were reduced < 50% by CTX GVIA 10-100 nM. 5 Capsaicin (3 muM) did not affect the CTX GVIA-sensitive or CTX GVIA-resistant neurogenic relaxations of the uterine artery. 6 The novel N-type blocker CTX CVID (100-300 nM), P/Q-type blockers agatoxin IVA (10-100 nM) or CTX CVIB (100 nM), the L-type blocker nifedipine (10 muM) or the 'R-type' blocker SNX-482 (100 nM), all failed to reduce CTX GVIA-resistant relaxations. The T-type channel blocker NiCl2 (100-300 muM) reduced but did not abolish the remaining neurogenic dilations. 7 Release of different neurotransmitters from the same autonomic vasomotor axon depends on similar subtypes of calcium channels. N-type channels are responsible for transmitter release from vasoconstrictor neurons innervating a muscular artery and capacitance vein, but only partly mediate release of nitric oxide and neuropeptides from pelvic vasodilator neurons.

Overexpressed Ca-v beta 3 inhibits N-type (Ca(v)2.2) calcium channel currents through a hyperpolarizing shift of "ultra-slow" and "closed-state" inactivation

It has been shown that P auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a hovel inhibitory effect of beta3 Subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/ cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at physiological holding potentials (HPs) of -60 and -80 mV At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca,.2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced Current suppression was reversed at a HP of - 120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba-2divided by (40 mM) as a charge carrier also largely diminished the effect of P3 at -80 mV Therefore, experimental conditions (HP, divalent cation concentration, and P3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel P3 effect. Steady-state inactivation curves revealed that N-type channels exhibited closed-state inactivation without P3, and that P3 caused an similar to40 mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of P3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV Similar ultra-slow inactivation was observed for N-type channels Without P3. Thus, P3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by Causing a hyperpolarizing shift of the inactivation without affecting ultra-slow and closed-state inactivation properties.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)

Introduction: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). Methods and materials: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells’ location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells’ VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium GreenTM-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. Results: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K+. This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. Conclusion: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

Structure-activity relationships of omega-conotoxins MVIIA, MVIIC and 14 loop splice hybrids at N and P/Q-type calcium channels

The omega-conotoxins are a set of structurally related, four-loop, six cysteine containing peptides, that have a range of selectivities for different subtypes of the voltage-sensitive calcium channel (VSCC). To investigate the basis of the selectivity displayed by these peptides, we have studied the binding affinities of two naturally occurring omega-conotoxins, MVIIA and MVIIC and a series of 14 MVIIA/MVIIC loop hybrids using radioligand binding assays for N and P/Q-type Ca2+ channels in rat brain tissue. A selectivity profile was developed from the ratio of relative potencies at N-type VSCCs (using [I-125]GVIA radioligand binding assays) and P/Q-type VSCCs (using [I-125]MVIIC radioligand binding assays). in these peptides, loops 2 and 4 make the greatest contribution to VSCC subtype selectivity, while the effects of loops 1 and 3 are negligible. Peptides with homogenous combinations of loop 2 and 4 display clear selectivity preferences, while those with heterogeneous combinations of loops 2 and 4 are less discriminatory. H-1 NMR spectroscopy revealed that the global folds of MVIIA, MVIIC and the 14 loop hybrid peptides were similar; however, several differences in local structure were identified. Based on the binding data and the 3D structures of MVIIA, GVIA and MVIIC, we have developed a preliminary pharmacophore based on the omega-conotoxin residues most Likely to interact with the N-type VSCC. (C) 1999 Academic Press.

Alterations in cardiac dihydropyridine receptors and calcium channel function in mdx mice

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular condition affecting approximately one in 3500 live male births resulting from the lack of the myocyte protein dystrophin. The absence of dystrophin in cardiac myocytes is associated with calcium overload which in turn activates calcium-dependent proteolytic enzymes contributing to congestive heart failure, muscle necrosis and fibrosis. To date, the basis for the calcium overload has not been determined. Since L-type calcium channels are a major mediator of calcium influx we determined their potential contribution to the calcium overload. Male muscular dystrophy (mdx) mice and control C57BL10ScSn (C57) mice aged 12– 16 weeks were used in all experiments. In tissue bath studies, isolated contracting left atria from mdx revealed a reduced potency to the dihydropyridine (DHP) agonist BayK8644 and antagonist nifedipine (P < 0.05). Similarly, radioligand binding studies using the DHP antagonist [3H]-PN 200-110 showed a reduced potency (P < 0.05) in isolated membranes, associated with an increased receptor density (P < 0.05). The increased receptor density was supported by RT-PCR experiments revealing increased RNAfor the DHP receptor. Patch clamp studies revealed the presence of a diltiazem sensitive calcium current that showed delayed inactivation in isolated mdx myocytes (P < 0.01). In conclusion, the increased number of DHP binding sites and the delay in L-type current inactivation may both contribute to increased calcium influx and hence calcium overload in the dystrophin deficient mdx cardiac myocytes.

Association between tumoral GH-releasing peptide receptor type 1a mRNA expression and in vivo response to GH-releasing peptide-6 in ACTH-dependent Cushing`s syndrome patients

Objective: GH secretagogues (GHS) produce exaggerated ACTH and cortisol responses in Cushing`s disease (CD) patients, attributable to their direct action on GH-releasing peptide receptor type la (GHSR-1a). However, there are no studies correlating the ill vivo response to GHS and GHSR-1a mRNA expression in ACTH-dependent Cushing`s syndrome (CS) patients. The aim of this study is to correlate the patterns of ACTH and cortisol response to GH-releasing peptide-6 (GHRP-6) to GHSR-1a expression in ACTH-dependent CS patients Design: Prospective study in a tertiary referral hospital center. Fifteen CD patients and two ectopic ACTH syndrome (EAS) patients were studied. Methods: Tumor fragments were submitted to RNA extraction, and GHSR-1a expression was studied through real-time qPCR and compared with normal tissue samples. The patients were also submitted to desmopressin test and vasopressin receptor type 1B (AVPR1B) mRNA analysis by qPCR. Results: GHSR-1a expression was similar in normal pituitary samples and in corticotrophic tumor samples. GHSR-1a expression was higher in patients (CD and EAS) presenting ill vivo response to GHRP-6. Higher expression of AVPR1B was observed in the EAS patients responsive to desmopressin, as well as in corticotrophic tumors, as compared with normal pituitary samples, but no correlation between AVPR1B expression and response to desmopressin was observed in the CD patients. Conclusions: Our results revealed a higher expression of GHSR-1a in the ACTH-dependent CS patients responsive to GHRP-6, suggesting an association between receptor gene expression and ill vivo response to the secretagogue in both the CD and the EAS patients.

Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)]; transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20 mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH. (c) 2011 Elsevier Ltd. All rights reserved.