Cardiac Calcium Channels Research Articles

Highly efficient synthesis of [11C]S12968 and [11C]S12967, for the in vivo imaging of the cardiac calcium channels using PET

AbstractThe dihydrophyridines S12968 ((−)‐S11568, absolute configuration S) and S12967 ((+)‐S11568, absolute configuration R), 3‐ethyl 5‐methyl (−/+)‐2‐[(2‐(2‐aminoethoxy)ethoxy)methyl]‐4‐(2,3‐dichlorophenyl)‐6‐methyl‐1,4‐dihydropyridine‐3,5‐dicarboxylate, have both an in vitro profile of high potency and of high selectivity for the low‐voltage‐dependent L‐type calcium channel. In this paper, the radiosynthesis of both enantiomers, S12968 and S12967, with carbon‐11, a positron‐emitting isotope (half‐life : 20.4 min) was investigated and oriented towards the preparation of multi milliCuries of radiotracer. Typically, 130–250 mCi (4.81–9.25 GBq) of [11C]S12968 and [11C]S12967 were obtained within 30 min of radiosynthesis (HPLC purification included) with specific radioactivities ranging from 500 to 1000 mCi/μmol (18.5–37.0 GBq/μmol) using no‐carrier‐added [11C]methyl triflate as the alkylating agent and the appropriate, enantiomerically pure carboxylic acid precursor at 100°C for 1 min. Based on preliminary PET experiments, only the levo enantiomer S12968 ((−)‐[11C]‐1) appears to be suitable for myocardial PET imaging as demonstrated in vivo in beagle dogs: with S12968, 85% of the uptake of [11C]S12968 could be inhibited in pretreatment experiments and up to 70% of [11C]S12968 could be displaced. Further investigations are currently underway in order to provide an absolute quantification of ventricular calcium channels with PET. Copyright © 2001 John Wiley & Sons, Ltd.

Synthesis of [11C]S12968 and [11C]S12967 for imaging the cardiac calcium channels using pet

AbstractThe dihydropyridines S12968 and S12967, two potent and selective calcium channel antagonists, have been labelled with carbon‐11 using no‐carrier‐added [11C]methyl triflate and the dedicated, enantiomerically pure carboxylic acid precursor at 100°C for 1 minute. Based on preliminary PET experiments, only S12968 appears to be suitable for myocardial PET imaging.

Negative inotropic effect of diazepam in isolated guinea pig heart.

The inotropic effect of diazepam, a benzodiazepine derivative, and its mechanism of action were examined using guinea pig heart and single ventricular cell preparations. In Langendorff hearts and right ventricular free-wall preparations, diazepam (10 to 100 microM) produced a monophasic negative inotropic effect in a concentration dependent manner. Neither a central type (flumazenil 1 microM) nor a peripheral type (PK11195 10 microM) of benzodiazepine receptor antagonist antagonized the monophasic negative inotropic effects of diazepam. Diazepam (10 to 100 microM) shortened action potential duration of papillary muscle in a concentration dependent manner. In isolated single ventricular cells, diazepam (30 and 100 microM) inhibited the calcium current (I(Ca)) in a concentration dependent manner. Diazepam produced a significant decrease in I(Ca) elicited by first depolarizing pulses, however, the decrease of I(Ca) was not augmented during a train of depolarizing pulses. Thus, diazepam appears to produce a tonic block of cardiac calcium channels and the mode of inhibition is clearly different from the use-dependent block of verapamil. From these results, it was concluded that diazepam produces a monophasic negative inotropic effect that is independent of the benzodiazepine receptor, and is probably mediated through an inhibition of I(Ca) in guinea pig heart preparations.

G αi2, G αi3and G αoare all Required for Normal Muscarinic Inhibition of the Cardiac Calcium Channels in Nodal/Atrial-like Cultured Cardiocytes

The cardiac l -type calcium current (ICa,L) is an important regulator of myocardial contractility. It is activated by sympathetic stimulation and inhibited by parasympathetic activity via muscarinic acetylcholine receptors. Muscarinic inhibition of ICa,Loccurs via activation of pertussis toxin (PTX)-sensitive heterotrimeric G-proteins. Although recent studies have shown that expression of Goαis important for this effect in adult mouse ventricular cells, two other PTX-sensitive G-proteins (Gi2and Gi3) are also expressed in cardiocytes and are activated. Their role in the regulation of ICa,Lhas not been examined. In addition, it is not known whether nodal/atrial cardiac cells use the same G-proteins. We show that gene inactivation of each of the three PTX-sensitive Gα-proteins (αi2, αi3, and αo) affects muscarinic inhibition of cardiac ICa,Lin embryonic stem (ES) cell-derived cardiocytes. Inactivation of eitherαi2 or αi3markedly slows the time course of muscarinic inhibition of ICa,L, and in cells where both αi2and αi3are inactivated the effects are not additive. We also establish an essential role for αoin this atrial/nodal-like cardiocyte system and show that αoacts proximal to NO generation. NO generation plays a critical role in ICa,Lregulation since the nitric oxide synthase (NOS) antagonist, l -NMMA, blocked the inhibition of ICa,Lin WT and in αi2/αi3-null cells. In WT cells, the NO generating agent SIN-1 inhibited ICa,Land the addition of carbachol resulted in faster inhibition, suggesting that pathways in addition to NO are also activated. This study shows that αi2and αi3play a critical role in the normal inhibition of cardiocyte ICa,L. Thus, all muscarinic receptor activated G-proteins (Gi2, Gi3and Go) are necessary for normal inhibition and act through both NO and non-NO signaling pathways.

Block and modified gating of cardiac calcium channel currents by terodiline.

1. Terodiline, an anticholinergic/antispasmodic drug effective in the treatment of urinary incontinence, is presently restricted due to adverse side effects on cardiac function. To characterize its effects on cardiac L-type Ca2+-channel current carried by Ca2+ (ICa, L) and Ba2+ (IBa,L), concentrations ranging from 0.1 to 100 microM were applied to whole-cell-configured guinea-pig ventricular myocytes. 2. Although sub-micromolar concentrations of terodiline had no effect on ICa,L at 0 mV, 100 microM drug reduced its amplitude to ca. 10% of pre-drug control. The estimated IC50 (15.2 microM in K+-dialysed cells, 12.2 microM in Cs+-dialysed cells; 0.1 Hz pulsing rate) is eight times higher than reported for ICa,L in bladder smooth muscle myocytes. 3. Terodiline affected ICa,L in a use-dependent manner; block increased when the pulsing rate was increased from 0.1 to 2 - 3 Hz, and when holding potential was lowered from -43 mV. The drug accelerated the decay of ICa,L at 0 mV in a concentration-dependent manner, and slowed the recovery of channels from inactivation. 4. Terodiline reduced peak IBa,L more effectively than peak ICa,L, and markedly accelerated the rate of inactivation of the current. 5. The results are discussed in terms of mechanisms of Ca2+ channel block and relation to the therapeutic and cardiotoxic effects of the drug.

Role of genomic mechanisms on cAMP-dependent positive inotropism in isolated left atrium of rat

It is well known that β-adrenoceptor stimulation induces positive inotropism by cAMP-dependent phosphorylation of cardiac calcium channels. Furthermore, hypertrophy of different tissues including the heart have been related to the stimulation of these adrenoceptors via mechanisms coupled to activation of transcription and protein synthesis. Early effects of isoproterenol mediated via this pathway has also been associated to the stimulation of β-adrenoceptors. However, the effects on the inotropism through genomic mechanisms have not yet been described. Isoproterenol (3 nM to 3 μM) induced a concentration-dependent positive inotropism, in isolated left atrium of male Wistar rats electrically stimulated (0.5 Hz, 5 ms, 30–50% above the threshold voltage), which was antagonized by atenolol (1 μM) and inhibited by a protein kinase A inhibitor, (R) p-cAMPS (10 μM). The inhibitor of transcription, actinomycin D (4 μM), and the protein synthesis inhibitor, cycloheximide (35.5 μM), significantly decreased the positive inotropism induced by isoproterenol. Forskolin (0.1 to 3 μM), an activator of adenylyl cyclase, induced a concentration-dependent positive inotropism which was also inhibited by (R) p-cAMPS, actinomycin D and cycloheximide. In the left atrium of rat, isoproterenol induced a positive inotropism which seems, at least in part, dependent upon intact transcription and protein synthesis, as suggested by the fact that the response was inhibited by the incubation with actinomycin D and cycloheximide. In addition, this genomic effect seems to be mediated by a cAMP-dependent mechanism. As it was inhibited by a protein kinase A inhibitor ((R) p-cAMPS) and similarly to isoproterenol, the positive inotropism induced by forskolin, which increases cytosolic cAMP, was also inhibited by actinomycin D and cycloheximide.

A Region in IVS5 of the Human Cardiac L-type Calcium Channel Is Required for the Use-dependent Block by Phenylalkylamines and Benzothiazepines

Mutations in motif IVS5 and IVS6 of the human cardiac calcium channel were made using homologous residues from the rat brain sodium channel 2a. [3H]PN200-110 and allosteric binding assays revealed that the dihydropyridine and benzothiazepine receptor sites maintained normal coupling in the chimeric mutant channels. Whole cell voltage clamp recording from Xenopus oocytes showed a dramatically slowed inactivation and a complete loss of use-dependent block for mutations in the cytoplasmic connecting link to IVS5 (HHT-5371) and in IVS5 transmembrane segment (HHT-5411) with both diltiazem and verapamil. However, the use-dependent block by isradipine was retained by these two mutants. For mutants HHT-5411 and HHT-5371, the residual current appeared associated with a loss of voltage dependence in the rate of inactivation indicating a destabilization of the inactivated state. Furthermore, both HHT-5371 and -5411 recovered from inactivation significantly faster after drug block than that of the wild type channel. Our data demonstrate that accelerated recovery of HHT-5371 and HHT-5411 decreased accumulation of these channels in inactivation during pulse trains and suggest a close link between inactivation gating of the channel and use-dependent block by phenylalkylamines and benzothiazepines and provide evidence of a role for the transmembrane and cytoplasmic regions of IVS5 in the use-dependent block by diltiazem and verapamil.

Synthesis and Characterization of a 11C-Labelled Derivative of S12968: An Attempt to Image In Vivo Brain Calcium Channels

[ 11C]S11568 (3-ethyl 5-methyl 2-[2-(2-aminoethoxy)ethoxymethyl]-4-(2,3-dichlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate) is a powerful ligand for the visualization of the cardiac calcium channel in vivo using PET. The aim of the present study was to synthesize a lipophilic, nonionized derivative of S11568 to facilitate its penetration into the brain. To increase the lipophilicity and to remove simultaneously the ionic nature of our ligand, the N-tert-butoxycarbonyl ( N-Boc) derivative of S11568 was synthesized. An IC 50 value of 1.7 nM for this derivative confirmed that both the affinity and selectivity for the calcium channel was unaltered by this chemical modification (S11568 with IC 50 value of 9.9 nM). The biologically more active enantiomer of S11568, the levogyre isomer S12968, was labelled with 11C using [ 11C]iodomethane. The lipophilicity of the N-Boc derivative was increased by a factor of three to four when compared to the parent compound (as determined by the measurement of the octanol/buffer partition coefficients). In vivo, this derivative slightly crosses the blood–brain barrier, as demonstrated by a 4-fold increase (with respect to the parent compound S12968) of the radioactivity in the brain using the 11C-labelled N-Boc S12968. This uptake remained too low to be suitable for imaging calcium channels.

Facilitation by the beta2a subunit of pore openings in cardiac Ca2+ channels.

1. Single channel recordings were performed on the cardiac calcium channel (alpha1C) in order to study the effect of coexpression of the accessory beta2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes. 2. The alpha1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states. 3. Coexpression of the beta2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the beta2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the beta2a subunit was found when the channel was gating in the low Po mode. 4. Slow gating transitions were also affected by the beta2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the beta2a subunit; while the alpha1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep. 5. The beta2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the alpha1C subunit has a tendency toward non-random mode switching. The beta2a subunit increased this tendency. A chi2 analysis of contingency tables indicated that the beta2a subunit caused the alpha1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep. 6. We conclude that the beta2a subunit causes changes to the alpha1C channel in both its fast and slow gating behaviour. The beta2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the beta2a subunit decreases the slow switching between low and high Po modes.

CAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels.

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac alpha1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the alpha1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.

Diazepam-induced Ca(2+)-channel blockade reduces hypothermia-induced electromechanical changes in isolated guinea pig ventricular muscle.

Calcium-channel blockers reduce the in vitro effects of hypothermia and benzodiazepines have been reported to reduce inward calcium flow through L-type cardiac-calcium channels. Thus, this study was designed to determine if diazepam could reduce hypothermia-induced changes in ventricular papillary muscle electromechanical activity. Conventional microelectrode techniques were used while force was recorded using a miniature force transducer. Six experimental groups of electrically paced papillary muscles were formed (n = 6 per group). One was exposed to one microM nisoldipine and four were exposed to one of four diazepam concentrations (0.1, 1.0, 10 or 100 microM). A final group had no drug and provided a time-matched control. The effects were determined at 37 degrees C and then at 27 degrees C. At 37 degrees C, diazepam initially increased and then reduced inotropy and APD90. Nisoldipine reduced both APD90 and inotropy. At 27 degrees C, 100 microM diazepam and nisoldipine (1.0 microM) reduced the hypothermia-induced lengthening of APD and the increase in force. Although diazepam reduced the hypothermia-induced alterations, the concentration required to do so (100 microM) suggests that this effect has little role in clinical use.

Diazepam-induced Ca2+-channel blockade reduces hypothermia-induced electromechanical changes in isolated guinea pig ventricular muscle

Calcium-channel blockers reduce thein vitro effects of hypothermia and benzodiazepines have been reported to reduce inward calcium flow through L-type cardiac-calcium channels. Thus, this study was designed to determine if diazepam could reduce hypothermia-induced changes in ventricular papillary muscle electromechanical activity. Conventional microelectrode techniques were used while force was recorded using a miniature force transducer. Six experimental groups of electrically paced papillary muscles were formed (n=6 per group). One was exposed to one μM nisoldipine and four were exposed to one of four diazepam concentrations (0.1, 1.0, 10 or 100 μM). A final group had no drug and provided a time-matched control. The effects were determined at 37°C and then at 27°C. At 37°C, diazepam initially increased and then reduced inotropy and APD90. Nisoldipine reduced both APD90 and inotropy. At 27°C, 100 μM diazepam and nisoldipine (1.0 μM) reduced the hypothermia-induced lengthening of APD and the increase in force. Although diazepam reduced the hypothermia-induced alterations, the concentration required to do so (100 μM) suggests that this effect has-little role in clinical use.

Molecular Studies on the Voltage Dependence of Dihydropyridine Action on L-type Ca2+ Channels

The interaction site(s) of dihydropyridine (DHP) antagonists and agonists have been identified by site-directed mutagenesis and localized on motifs IIIS5, IIIS6, and IVS6 of L-type voltage-gated calcium channels. In this study, we investigated the voltage-dependent action of DHPs with mutants of the IIIS6 and IVS6 segments of a cardiac calcium channel. Tyrosine residues in both motifs (Tyr1178 and Tyr1489) strongly contributed to the action of DHP agonists and antagonists. When these two sites were mutated, the communication between the voltage sensor and the DHP interaction site(s) was substantially impaired. In contrast, mutants of a nearby Ile (Ile1182) had much less influence on DHP agonist and antagonist interaction, and the voltage dependence of DHP antagonists was very similar to that of the wild type. The effect of a mutating of Ile1182, on agonist or antagonist action, however, depended strongly on the type of amino acid change. When Ile1182 was substituted with alanine, small changes were noted for DHP agonist and antagonist action. Changing this site into phenylalanine, however, significantly decreased the action of the DHP antagonist. These data show that Ile1182 can preferentially interact with DHP antagonists, but has a lesser contribution in agonist interaction. Thus, even though the agonist and antagonist interaction sites for DHPs with L-type calcium channels may overlap, some amino acids in this site may exhibit a preference for either DHP enantiomers.

Identification and functional characterization of a calcium channel gamma subunit.

A positive selection technique was used to identify novel auxiliary calcium channel subunits that are similar to the skeletal muscle gamma subunit. A new rat gamma subunit cDNA was found, which was highly expressed in skeletal muscle tissue and was detected by RT-PCR in cardiac tissue. The 223-amino-acid-protein shares 84% and 79% identity, respectively, with the human and rabbit skeletal muscle subunits. Northern blot analysis revealed a single transcript of 1.5 kb in rat skeletal muscle, but not in cardiac tissue. Transient coexpression with the cardiac calcium channel complex demonstrated that the gamma subunit shifted the inactivation curve to negative potentials and accelerated current inactivation without changing other voltage-dependent properties of the channel.

Inhibition of Cloned Human L-Type Cardiac Calcium Channels by 2,3-Butanedione Monoxime Does Not Require PKA-Dependent Phosphorylation Sites

The oxime derivative 2,3-butanedione monoxime (BDM) is used as an inorganic phosphatase to probe the phosphorylation state of many cellular proteins including the L-type calcium channel in various tissues. We used BDM further to shed light on the controversy surrounding direct phosphorylation of the L-type Ca2+channel. We employed a recombinant system that utilizes HEK 293 cells expressing wild type and mutant human heart calcium channels. BDM reversibly reduced the calcium channel current induced by expression of the wild type channel in a concentration-dependent manner with an apparent IC50value of 15.3 mM. Deletion of part of the carboxyl terminus of the α1subunit, which contains one putative protein kinase A site, or mutating all of the protein kinase A consensus sites of the pore forming subunit, did not significantly change the apparent IC50value or alter in any other way the blocking effect of BDM on the expressed currents. Our data suggest that BDM produces reversible modifications of the cardiac calcium channel protein leading to an expected reduction in the amplitude of the expressed currents, but the site of action must be different from that of the consensus sites for protein kinase A dependent phosphorylation.

Charged amino acids near the pore entrance influence ion-conduction of a human L-type cardiac calcium channel.

Voltage-dependent L-type Ca2+ channels form highly selective pores for Ca2+ ions in the membranes of excitable cells. We investigated the functional role of negatively charged residues, within or near the selectivity region, in ion permeation of a human cardiac L-type Ca2+ channel. Glutamates in each of the four repeats, and an aspartate in repeat IV, were substituted with positively charged lysine. Wild-type and mutant Ca2+ channels were expressed in Xenopus oocytes. Block by Ca2+ and Mg2 of inward Li+ currents through the channels was used to assess the effects of amino acid substitutions on high-affinity divalent cation binding. The rank order of IC50's for Ca2+ block of I(Li) was: E677K > E1086K > E334K > E1387K > D1391K > or approximately wild-type. The order of IC50's for Mg2+ block of I(Li) indicated differential involvement of the same residues in Mg2+ binding: E 1387K > E334K > E1086K > E677K > D 1391K = wild-type. Mutants E1387K and D1391K effectively permeated Ba2+, but exhibited a decreased single-channel conductance. The unitary current amplitude carried by Na+, in the absence of external divalent cations, was slightly decreased in the E1387K mutant but not in the D1391K mutant. The results confirm that each of the four glutamates participate unequally in high-affinity Ca2+ binding. Additionally, our results indicate that these glutamate residues participate in Mg2+ binding. The glutamate at position 1387 may be only peripherally involved in the formation of a high-affinity Ca2+ -binding site but is central to a Mg2+ binding site accessible from the external side of the pore. The aspartate at position 1391 is most likely located just external to the selectivity region.

Depolarization shifts the voltage dependence of cardiac sodium channel and calcium channel gating charge movements

Depolarization shifts the voltage dependence of cardiac sodium channel and calcium channel gating charge movements

Physiologic and emerging pathophysiologic role of cardiac calcium channels

The link between cardiac contractile dysfunction in patients with end-stage heart failure and aberrant myocardial intracellular calcium handling is now well established. The precise intracellular protein(s) responsible for this breakdown in calcium handling is at present unclear. However, a number of distinct sarcolemmal (L-type, N-type, T-type, P-type, Q-type) and sarcoplasmic reticular (calcium release, ryanodine) calcium channels that have been defined on a biophysical, biochemical, and molecular basis lend valuable insights into possible factors that may contribute to the abnormal calcium handling in the hearts of these patients. What is now clear is that cardiac muscle contraction is a rigorously regulated event that follows the organized cycling of calcium from the sarcoplasmic reticulum (SR) into the cytosol and back into the SR, and that this cycle follows the graded entry of trigger calcium that enters the cell through the voltage-sensitive calcium channel. Furthermore, the voltage-dependent properties of potential-dependent calcium channels provide the underpinning for the vascular selectivity of the clinically available calcium channel drugs. Moreover, it has also been reported that the efficacy of these agents is augmented in pathologic (ischemic) tissue owing to the state dependence of these channels. Recently, the basis for the critical role of the SR in calcium signaling has started to emerge. The SR calcium handling proteins (SR calcium release channel/ryanodine receptor, SR Ca2+ ATPase, phospholamban, calsequestrin) play a critical role in maintaining intracellular free ionized calcium concentrations ([Ca2+i), which therefore regulate systolic and diastolic function on a beat-to-beat basis within the cardiac cell. This rigorous control of [Ca2+]i is in part the result of the highly developed junctional regions of the cardiac SR. Elucidation of the calcium handling process in these regions and the potential damage resulting from cardiovascular disease has been greatly aided by the invaluable molecular tool, ryandine. From an expanding volume of information provided by animal models of ischemia, hypertrophy, and heart failure, it now appears that changes in cardiac voltage-sensitive calcium channels are likely to be the result of a secondary process that may not be directly linked to the onset of these cardiovascular diseases. Conversely, the regulation of ryanodine receptors has been suggested to be a mechanism initiating the decline in myocardial contractility leading to heart failure. These reports have been supported by studies demonstrating SR calcium release channel/ryanodine receptor changes in pressure overload hypertrophy and myocardial ischemia. Further support for the role of SR calcium release channels in cardiovascular disease is found in reports that couple leaking SR channels with ischemia and cardiac failure. These results suggest that changes in SR calcium handling proteins may be critically linked to cardiovascular disease. A more central question stemming from these results is exactly how these altered SR calcium handling proteins are involved with the onset and progression of cardiovascular disease. Application of transgenic technologies and animal models of chronic heart failure that parallel the human condition will provide the means necessary for unequivocally determining if the apparent adaptive changes in calcium handling are associated with the onset and progression of this syndrome.

Lack of Involvement of Protein Kinase A Phosphorylation in Voltage-Dependent Facilitation of the Activity of Human Cardiac L-Type Calcium Channels

Phosphorylation by protein kinase A is thought to be involved in voltage-dependent facilitation of calcium channels. Here we have shown that the subunit complex of a cloned human cardiac calcium channel, expressed inXenopusoocytes, responds to voltage-dependent facilitation by an approximately 50% increase of the calcium channel peak current. The removal of all protein kinase A consensus sequences by site-directed mutagenesis decreased but did not eliminate the response to prepulse facilitation. Moreover, Rp-cAMP-S, an inhibitor of protein kinase A, could not prevent facilitation of the wild-type calcium channel currents. Similarly, AMP-PNP a nonhydrolyzable analog of ATP, while significantly decreasing the whole-cell current amplitude, failed to reduce the response to double-pulse facilitation. Therefore, we conclude that the voltage-dependent facilitation of cloned calcium channel currents is not due to enhancement of phosphorylation, but probably to some type of voltage-induced conformational change in the channel.

Regulation of calcium channel expression in neonatal myocytes by catecholamines.

Expression of the dihydropyridine (DHP) receptor (alpha 1 subunit of L-type calcium channel) in heart is regulated by differentiation and innervation and is altered in congestive heart failure. We examined the transmembrane signaling pathways by which norepinephrine regulates DHP receptor expression in cultured neonatal rat ventricular myocytes. Using a 1.3-kb rat cardiac DHP receptor probe, and Northern analysis quantified by laser densitometry, we found that norepinephrine exposure produced a 2.2-fold increase in DHP receptor mRNA levels at 2 h followed by a decline to 50% of control at 4-48 h (P < 0.02). The alpha-adrenergic agonist phenylephrine and a phorbol ester produced a decline in mRNA levels (8-48 h). The beta-adrenergic agonist isoproterenol and 8-bromo-cAMP produced a transient increase in mRNA levels. After 24 h of exposure to isoproterenol, 3H-(+)PN200-110 binding sites increased from 410 +/- 8 to 539 +/- 39 fmol/mg (P < 0.05). The number of functional calcium channels, estimated by whole-cell voltage clamp experiments, was also increased after 24 h of exposure to isoproterenol. Peak current density (recordings performed in absence of isoproterenol) increased from -10.8 +/- 0.8 (n = 23) to -13.9 +/- 1.0 pA/pF (n = 27) (P < 0.01). Other characteristics of the calcium current (voltage for peak current, activation, and inactivation) were unchanged. Exposure for 48 h to phenylephrine produced a significant decline in peak current density (P < 0.01). We conclude that beta -adrenergic transmembrane signaling increases DHP receptor mRNA and number of functional calcium channels and that alpha - adrenergic transmembrane signaling produces a reciprocal effect. Regulation of cardiac calcium channel expression by adrenergic pathways may have physiological and pathophysiological importance.