Role Of L-type Calcium Channels Research Articles

Role of L-type calcium channels in pacing-induced short-term and long-term cardiac memory in canine heart.

We tested the hypothesis that ICa,L is important to the development of cardiac memory. The effects of L-type Ca2+ channel blockade and beta-blockade were tested on acutely anesthetized and on chronically instrumented, conscious dogs. Short-term memory (STM) was induced by 2 hours of ventricular pacing and long-term memory (LTM) by ventricular pacing for 21 days. STM dogs received placebo, nifedipine, or propranolol, and LTM dogs received placebo, atenolol, or amlodipine. AT1 receptor blockade (candesartan) and ACE inhibition (trandolapril) were also tested in LTM. Microelectrodes were used to record transmembrane potentials from isolated epicardial and endocardial slabs using a protocol simulating STM in intact animals. Left ventricular epicardial myocytes from LTM or sham control dogs were dissociated, and ICa,L was recorded (whole-cell patch-clamp technique). Evolution of STM and LTM was attenuated by ICa,L blockers but not beta-blockers. Neither AT1 receptor blockade nor ACE inhibition suppressed LTM. In microelectrode experiments, pacing induced an epicardial-endocardial gradient change mimicking STM that was suppressed by nifedipine. In patch-clamp experiments, peak ICa,L density in LTM and control were equivalent, but activation was more positive and time constants of inactivation longer in LTM (P<0.05). ICa,L blockade but not beta-adrenergic blockade suppresses cardiac memory. LTM evolution is unaffected by angiotensin II blockade and is associated with altered ICa,L kinetics.

Coexpression of a Ca v1.2 protein lacking an N-terminus and the first domain specifically suppresses L-type calcium channel activity

L-type Ca 2 channels play a critical role in many types of cells, including nerve, muscle and endocrine cells. The most popular and effective tools for analyzing the roles of L-type calcium channels (L-channels) are specific antagonists such as dihydropyrigines. With these drugs however, it is difficult to target specific cells. One solution is to develop a genetically targetable inhibitor coded by DNA. As a candidate for such an inhibitor, a dominant negative mutant of Ca v1.2 was designed by mimicking an ascidian 3-domain-type α1 subunit (that inhibits the full-length subunit’s current). The 3-domain-type Ca v1.2 subunit significantly inhibited wild-type Ca v1.2 current, but not other ionic currents such as Ca v2.1 and Na v channels in Xenopus oocyte expression systems. Western blot analysis showed that the expression of the wild-type protein into the plasma membrane was significantly suppressed on coexpression with the truncated protein. These findings support that an N-terminus-truncated mutant could serve as a specific genetically encoded inhibitor for L-channels.

P2 receptor-mediated afferent arteriolar vasoconstriction during calcium blockade.

Experiments were performed to determine the role of L-type calcium channels on the afferent arteriolar vasoconstrictor response to ATP and UTP. With the use of the blood-perfused juxtamedullary nephron technique, kidneys were perfused at 110 mmHg and the responses of arterioles to alpha,beta-methylene ATP, ATP, and UTP were determined before and during calcium channel blockade with diltiazem. alpha,beta-Methylene ATP (1.0 microM) decreased arteriolar diameter by 8 +/- 1% under control conditions. This response was abolished during calcium channel blockade. In contrast, 10 microM UTP reduced afferent arteriolar diameter to a similar degree before (20 +/- 4%) and during (14 +/- 4%) diltiazem treatment. Additionally, diltiazem completely prevented the vasoconstriction normally observed with ATP concentrations below 10 microM and attenuated the response obtained with 10 microM ATP. These data demonstrate that L-type calcium channels play a significant role in the vasoconstrictor influences of alpha,beta-methylene ATP and ATP but not UTP. The data also suggest that other calcium influx pathways may participate in the vasoconstrictor response evoked by P2 receptor activation. These observations support previous findings that UTP-mediated elevation of intracellular calcium concentration in preglomerular vascular smooth muscle cells relies primarily on calcium release from intracellular pools, whereas ATP-mediated responses involve both voltage-dependent calcium influx, through L-type calcium channels, and the release of calcium from intracellular stores. These results support the argument that P2X and P2Y receptors influence the diameter of afferent arterioles through activation of disparate signal transduction mechanisms.

Differential Involvement of L-Type Calcium Channels in Epileptogenesis of Rat Hippocampal Slices during Ontogenesis

Organic calcium channel antagonists block epileptiform activity in adult tissue, suggesting an essential role of L-type channels in epileptogenesis in the mature CNS. By contrast, this remains doubtful for neonatal tissue, as the density of calcium channels changes markedly with ontogenesis. The paper addresses this question by exploring the antiepileptic efficacy of the L-type calcium channel blockers verapamil and nifedipine in low-Mg2+-epilepsy in rat hippocampal slices of different postnatal (PN) ages. Field (CA3, CA1) and membrane potentials (CA3) were recorded. Washout of Mg2+ induced epileptiform potentials, which were blocked age-dependently: Verapamil suppressed activity in all preparations of PN1–5 and PN13–30+, but only in 70% of PN6–12. Nifedipine depressed activity in >75% of slices of PN13–30+, but only in 33% of PN1–12. The findings indicate a role of L-type calcium channels in epileptogenesis from PN13 onwards, with phenylalkylamine-sensitive calcium channels also being involved during PN1–5.

L-type calcium channels selectively control the defensive behaviors induced by electrical stimulation of dorsal periaqueductal gray and overlying collicular layers

The present study reports the involvement of l-type calcium channels in the control of defensive behaviors produced by electrical stimulation of dorsal periaqueductal gray and overlying collicular layers. Rats that had chemitrodes in the dorsal midbrain and which stimulation produced freezing or flight behaviors with less than 55 μA were selected for drug experiments. Stimulation was repeated the day after the screening session 20 min following the microinjection into the dorsal periaqueductal gray of 15 nmol of either verapamil, a selective l-type calcium channel antagonist, or cobalt chloride (CoCl 2), a calcium-specific channel modulator. Post-drug sessions were performed 48 h after. Threshold functions were obtained by logistic fitting of accumulated response frequencies. Verapamil and CoCl 2 significantly attenuated the output of immobility, exophthalmus, running and jumping. Although to a lesser degree, verapamil also attenuated defecation. Because CoCl 2 had no effect on defecation, the attenuation of this response by verapamil suggests a non-specific action of this drug. Neither verapamil nor CoCl 2 changed the output of micturition. Finally, whereas there was a complete recovery of defensive thresholds following the microinjection of verapamil, the attenuating effects of CoCl 2 were still present 48 h after. These results support an important role of l-type calcium channels in the neurogenesis of dorsal periaqueductal gray-evoked immobility, exophthalmus, running and jumping, but not defecation and micturition responses.

Calcium-channel activation and matrix protein upregulation in bone cells in response to mechanical strain

Femur-derived osteoblasts cultured from rat femora were loaded with Fluo-3 using the AM ester. A quantifiable stretch was applied and [Ca(2+)]i levels monitored by analysis of fluorescent images obtained using an inverted microscope and laser scanning confocal imaging system. Application of a single pulse of tensile strain via an expandable membrane resulted in immediate increase in [Ca(2+)]i in a proportion of the cells, followed by a slow and steady decrease to prestimulation levels. Application of parathyroid hormone (10(-6) M) prior to mechanical stimulation potentiated the load-induced elevation of [Ca(2+)]i. Mechanically stimulating osteoblasts in Ca(2+)-free media or in the presence of either nifedipine (10 microM; L-type Ca(2+)-channel blocker) or thapsigargin (1 microM; depletes intracellular Ca(2+) stores) reduced strain-induced increases in [Ca(2+) ]i. Furthermore, strain-induced increases in [Ca(2+)]i were enhanced in the presence of Bayer K 8644 (500 nm), an agonist of L-type calcium channels. The effects of mechanical strain with and without inhibitors and agonists are described on the total cell population and on single cell responses. Application of strain and strain in the presence of the calcium-channel agonist Bay K 8644 to periosteal-derived osteoblasts increased levels of the extracellular matrix proteins osteopontin and osteocalcin within 24 h postload. This mechanically induced increase in osteopontin and osteocalcin was inhibited by the addition of the calcium-channel antagonist, nifedipine. Our results suggest an important role for L-type calcium channels and a thapsigargin-sensitive component in early mechanical strain transduction pathways in osteoblasts.

Effects of nimodipine on extracellular dopamine levels in the rat nucleus accumbens in ethanol withdrawal

Withdrawal from chronic ethanol intoxication is associated with a reduction of dopamine neurotransmission. However, the mechanisms of dopamine depletion, a putative neurochemical correlate of the dysphoric symptomatology, are not yet understood. To assess the role of L-type calcium channels in the inhibition of the dopaminergic system in the withdrawal state, the effects of the dihydropyridine calcium channel antagonist nimodipine on the extracellular levels of dopamine were studied in the nucleus accumbens shell of awake rats 10 h after withdrawal from chronic ethanol intoxication. In control, chronic sucrose-withdrawn rats, nimodipine did not change extracellular dopamine levels. However, in ethanol-withdrawn rats nimodipine (5 or 10 mg/kg s.c.) increased extracellular dopamine to 136±16 and 305±19% of pre-administration values, respectively, the latter dose elevating levels above those of controls. The elevations of extracellular DA by nimodipine (10 mg/kg) were associated with a significant reduction (−17%) of the overall behavioural score of the withdrawal symptomatology, as evaluated for 11 behavioural items. Significant reductions of the score for convulsions (−47%) and, to a lesser extent, for catatonia (−30%) and tremors (−15%) contributed to the overall effect. It is suggested that overactivity of L-type calcium channels is involved in the mechanisms of dopamine depletion as well as in certain behavioural/neurological signs associated with ethanol withdrawal. By restoring depleted dopamine levels, dihydropyridines might ameliorate the dysphoric symptoms of ethanol abstinence.

Purinoceptor-mediated calcium signaling in preglomerular smooth muscle cells.

-The current studies were performed to determine the contribution of calcium mobilization and voltage-dependent calcium influx to the increase in [Ca2+]i elicited by ATP and UTP. Suspensions of freshly isolated smooth muscle cells were prepared from preglomerular microvessels by enzymatic digestion and loaded with the Ca2+-sensitive dye fura 2. The effect of ATP and UTP on [Ca2+]i was studied on single cells with standard microscope-based fluorescence photometry techniques. Resting [Ca2+]i averaged 80+/-3 nmol/L (n=219 single cells from 58 dispersions). ATP (100 micromol/L) increased [Ca2+]i to a peak value of 845+/-55 nmol/L (n=70 single cells from 38 dispersions) before stabilizing at 124+/-81 nmol/L. Similarly, 100 micromol/L UTP (n=39 single cells from 26 dispersions) stimulated a peak increase in [Ca2+]i of 1426+/-584 nmol/L before reaching a stable plateau of 123+/-10 nmol/L. The [Ca2+]i response to ATP and UTP was also assessed in the absence of extracellular calcium. In these studies, exposure to 100 micromol/L ATP induced a transient peak increase in [Ca2+]i, with the plateau phase being totally abolished. In contrast, exposure to 100 micromol/L UTP under calcium-free conditions resulted in no detectable change in the UTP-mediated increase in [Ca2+]i. The role of L-type calcium channels in the response was assessed with the calcium channel antagonist diltiazem. Incubation with diltiazem (10 micromol/L) markedly reduced the response to ATP, whereas the response to UTP was only slightly reduced. These data demonstrate that both ATP and UTP directly stimulate a biphasic increase in [Ca2+]i in renal microvascular smooth muscle cells. Furthermore, the data suggest that the elevation of [Ca2+]i elicited by ATP is largely dependent on calcium influx through L-type calcium channels, whereas the response to UTP appears to derive primarily from mobilization of calcium from intracellular stores.

Tyrosine Hydroxylase Expression in Primary Cultures of Olfactory Bulb: Role of L-Type Calcium Channels

Sensory activity mediates regulation of tyrosine hydroxylase (TH), the first enzyme in the dopamine biosynthetic pathway, in the rodent olfactory bulb. The current studies established for the first time primary cultures of neonatal mouse olfactory bulb expressing TH and tested whether L-type calcium channels mediate the activity-dependent regulation of the dopamine phenotype. After 1 d in vitro (DIV), a small population of TH-immunostained neurons that lacked extensive processes could be demonstrated. After an additional 2 DIV in serum-free medium, the number of TH neurons had doubled, and they exhibited long interdigitating processes. Membrane depolarization for 48 hr with 50 mM KCl produced a further 2.4-fold increase in the number of TH-immunoreactive neurons compared with control cultures. Increased TH neuron number required at least 36 hr of exposure to KCl. Forskolin, which increases intracellular cAMP levels, induced a 1.5- to 1.6-fold increase in the number of TH-immunostained neurons. Combined treatment with KCl and forskolin was not additive. Nifedipine, an L-type calcium channel blocker, completely prevented the depolarization-mediated increase in TH expression but did not block the response to forskolin. Treatment with Bay K8644, an L-type calcium channel agonist, also significantly increased the number of TH-expressing neurons. Depolarization also induced alterations in neuritic outgrowth, resulting in a stellate versus an elongate morphology that, in contrast, was not prevented by nifedipine. These results are the first demonstration that in vitro, as in vivo, depolarization increases TH expression in olfactory bulb and that L-type calcium channels mediate this activity-dependent regulation of the dopamine phenotype.

A Role for L-Type Calcium Channels in Developmental Regulation of Transmitter Phenotype in Primary Sensory Neurons

To examine the influence of activity-dependent cues on differentiation of primary afferent neurons, we investigated the short- and long-term effects of depolarization and calcium influx on expression of transmitter traits in sensory ganglion cell cultures. We focused on expression of tyrosine hydroxylase (TH), a marker for dopaminergic neurons, in developing petrosal ganglion (PG), nodose ganglion, and dorsal root ganglion neurons grown in the presence or absence of depolarizing concentrations of KCl. Exposure to 40 mM KCl increased the proportion of TH-immunoreactive neurons in all three ganglia in a developmentally regulated manner that corresponded to the temporal pattern of dopaminergic expression in vivo. PG neurons, for example, were most responsive to elevated KCl on embryonic day 16.5 (E16.5), the age at which the dopaminergic phenotype is first detectable in vivo. However, KCl was relatively ineffective at increasing TH expression in neonatal PG, indicating a critical period for induction of this phenotype by depolarization. Detailed analysis of TH induction in PG neurons demonstrated that, although N-type calcium channels carried the majority of the high voltage-activated calcium current, only L-type calcium channel blockade inhibited the effect of elevated KCl. Further studies revealed that after removal of high KCl, neurons remained sensitized to subsequent stimulation for >1 week. Specifically, cultures exposed to KCl beginning on E16.5 (the conditioning stimulus), then returned to control medium, and subsequently re-exposed to elevated KCl after 9 d (the test stimulus) contained fourfold more TH-positive neurons than did cultures exposed to the test stimulus alone. Moreover, blockade of L-type calcium channels during the conditioning stimulus completely abolished long-term potentiation of the TH response to elevated KCl. These findings demonstrate a novel role for L-type calcium channels in activity-dependent plasticity of transmitter expression in sensory neurons and indicate that exposure to depolarizing stimuli during early development may alter neuronal response properties at later ages.

Depolarization-stimulated cholecystokinin secretion is mediated by L-type calcium channels in STC-1 cells

To examine the role of calcium channels in depolarization-activated cholecystokinin (CCK) release, studies were performed in an intestinal CCK-secreting cell line, STC-1. Blockade of potassium channels with barium chloride (5 mM) increased the release of CCK by 374.6 +/- 46.6% of control levels. Barium-induced secretion was inhibited by the L-type calcium-channel blocker, nicardipine. Nicardipine (10(-9)-10(-5) M) produced a dose-dependent inhibition in barium-stimulated secretion with a half-maximal inhibition (IC50) value of 0.1 microM. A second L-type calcium-channel blocker, diltiazem (10(-9)-10(-4) M), also inhibited barium-induced CCK secretion with an IC50 value of 5.1 microM. By contrast, the T-type calcium-channel blocker, nickel chloride (10(-7)-10(-8) M), failed to significantly inhibit barium-induced CCK secretion. To further evaluate a role for L-type calcium channels in the secretion of CCK, the effects of the L-type calcium channel opener, BAY K 8644, were examined. BAY K 8644 (10(-8)-10(-4) M) produced a dose-dependent stimulation in CCK release with a mean effective concentration value of 0.2 microM. Recordings of single-channel currents from inside-out membrane patches showed activation of calcium channels by BAY K 8644 (1 microM), with a primary channel conductance of 26.0 +/- 1.2 pS. It is concluded that inhibition of potassium channel activity depolarizes the plasma membrane, thereby activating L-type, but not T-type, calcium channels. The corresponding influx of calcium serves to trigger secretion of CCK.

Role of L-Type Calcium Channels on Stimulated Calcium Influx and on Proliferative Activity of Human Coronary Smooth Muscle Cells

Summary: Dihydropyridine (DHP) calcium channel blockers are widely used in treatment of coronary artery disease. To evaluate the specific role of L-type calcium channels in the antianginal and possibly antiatherosclerotic properties of DHP inhibitors, we examined the effects of a 1,4-DHP agonist and antagonist on angiotensin II (ANG II)- and serum-stimulated calcium influx and proliferation of human coronary smooth muscle cells (cSMC). Fluorometry of fura-2 was used to measure changes in free cytosolic Ca2+ concentration ([Ca2+]i) in cSMC after short- and long-term pretreatment with the calcium agonist Bay K 8644 or the antagonist nitrendipine, respectively. Proliferative activity was quantified during exponential growth in serum-supplemented medium with or without both DHPs. Short- and long-term pretreatment with Bay K 8644 increased basal [Ca2+]i significantly in resting cells and augmented ANG II- and serum-induced sustained [Ca2+]i responses. Concordantly, proliferation rate was increased. In contrast, nitrendipine had no significant effect on basal or stimulated [Ca2+]i after short-term treatment, but decreased [Ca2+]i after 24-h incubation, attenuated the plateau phase of ANG II- and serum-evoked [Ca2+]i transients, and reduced proliferative activity of these cells. The results indicate that 1,4-DHPs modulate ANG II- and seruminduced Ca2+ influx in cSMC. Thus, L-type calcium channels may contribute to [Ca2+]i transients evoked by ANG II and serum. Moreover, the modulating effects of both DHPs on proliferative activity suggest involvement of DHP-sensitive calcium channels. Calcium influx through L-type channels may be one of the mechanisms that determine responsiveness to vasoconstrictors and proliferative activity of human cSMC.

Role of L-Type Calcium Channels on Stimulated Calcium Influx and on Proliferative Activity of Human Coronary Smooth Muscle Cells

Dihydropyridine (DHP) calcium channel blockers are widely used in treatment of coronary artery disease. To evaluate the specific role of L-type calcium channels in the antianginal and possibly antiatherosclerotic properties of DHP inhibitors, we examined the effects of a 1,4-DHP agonist and antagonist on angiotensin II (ANG II)- and serum-stimulated calcium influx and proliferation of human coronary smooth muscle cells (cSMC). Fluorometry of fura-2 was used to measure changes in free cytosolic Ca2+ concentration ([Ca2+]i) in cSMC after short- and long-term pretreatment with the calcium agonist Bay K 8644 or the antagonist nitrendipine, respectively. Proliferative activity was quantified during exponential growth in serum-supplemented medium with or without both DHPs. Short- and long-term pretreatment with Bay K 8644 increased basal [Ca2+]i significantly in resting cells and augmented ANG II- and serum-induced sustained [Ca2+]i responses. Concordantly, proliferation rate was increased. In contrast, nitrendipine had no significant effect on basal or stimulated [Ca2+]i after short-term treatment, but decreased [Ca2+]i after 24-h incubation, attenuated the plateau phase of ANG II- and serum-evoked [Ca2+]i transients, and reduced proliferative activity of these cells. The results indicate that 1,4-DHPs modulate ANG II- and serum-induced Ca2+ influx in cSMC. Thus, L-type calcium channels may contribute to [Ca2+]i transients evoked by ANG II and serum. Moreover, the modulating effects of both DHPs on proliferative activity suggest involvement of DHP-sensitive calcium channels. Calcium influx through L-type channels may be one of the mechanisms that determine responsiveness to vasoconstrictors and proliferative activity of human cSMC.