Calcium Channel Currents Research Articles

Differential expression of four Cav3.1 splice variants in the repeat iii‐iv loop

Molecular cloning revealed the three isoforms (Cav3.1, Cav3.2, and Cav3.3) of the T‐type calcium channel subfamily. Expression studies exhibited their distinctive electrophysiological and pharmacological properties, accounting for diverse properties of T‐type calcium channel currents previously characterized from isolated cells. However, electrophysiological properties of ion channels have shown to be more diversified by their splice variants. We here searched splice variants of rat Cav3.1 T‐type channel by reverse‐transcription‐polymerase chain reaction (RT‐PCR) to further explore diversity of Cav3.1. Interestingly, analyses of cloned RT‐PCR products displayed that there were at least four splicing variants of rat Cav3.1 in the loop connecting repeats III and IV. Southern blot analyses indicated that the predominantly detected variant in brain was Cav3.1a (492 bp), which were rarely detected in most of peripheral tissues. Other two variants (Cav3.1c, 546 bp; Cav3.1d, 525 bp) were detected in most of the tissues examined. The smallest isoform (Cav3.1b, 471 bp) was rarely detected all the tissues. Electrophysiological characterization of the splicing variants indicated that the splice variants differ in inactivation kinetics and the voltage dependence of activation and inactivation as well.

Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin

The mechanism of action of gabapentin is still not well understood. It binds to the α2δ-1 and α2δ-2 subunits of voltage-gated calcium channels but has little acute effect on calcium currents in several systems. However, our recent results conclusively demonstrated that gabapentin inhibited calcium currents when applied chronically but not acutely, both in heterologous expression systems and in dorsal root ganglion neurons 1. In that study we only examined a 40 hour time point of incubation with gabapentin, and here we have extended these results to include the effect of up to 6 and 20 hours incubation with gabapentin on calcium channel currents formed from CaV2.1/β4/α2δ-2 subunits. Gabapentin was significantly effective to inhibit the currents if included for 17-20 hours prior to recording, but it did not produce a significant inhibition if included for 3-6 hours. We previously concluded that gabapentin acts primarily at an intracellular location, requiring uptake into cells. However, this effect is mediated by α2δ subunits, being prevented by mutations in either α2δ-1 or α2δ-2 that abolish gabapentin binding 1. Furthermore, we also showed that the trafficking of α2δ-2 and CaV2 channels was disrupted by gabapentin. Here we have also extended that study, to show that the cell-surface expression of CaV2.1 is not reduced by chronic gabapentin if it is co-expressed with α2δ-2 containing a point mutation (R282A) that prevents gabapentin binding

Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes

Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K(+) current (I(K(ACh))) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by approximately 25% (vs. untreated controls) in field-stimulated myocytes. S1P(1) was shown to be involved by using the S1P(1)-selective agonist SEW2871 on myocytes isolated from S1P(3)-null mice. However, in these myocytes, S1P(3) can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P(1) antagonist VPC23019. Since S1P(1) activates G(i) exclusively, whereas S1P(3) activates both G(i) and G(q), these results strongly implicate the involvement of mainly G(i). Additional experiments using the I(K(ACh)) blocker tertiapin demonstrated that I(K(ACh)) can contribute to the negative inotropy following S1P activation of S1P(1) (perhaps through G(ibetagamma) subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by I(K(ACh))) can reduce L-type calcium current and thus can decrease the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via G(i) to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca(2+)](i); and 2) a second pathway that acts via G(i) to activate I(K(ACh)) and reduce APD. This decrease in APD is expected to decrease Ca(2+) influx and reduce [Ca(2+)](i) and myocyte contractility.

Cholinoreceptor Autoantibodies in Sjögren Syndrome

Previous studies have demonstrated that antibodies against cholinoreceptors of exocrine glands correlate with dry mouth in persons with primary Sjögren syndrome (pSS). The aim of the present investigation was to establish if serum IgG antibodies (pSS IgG) were able to interact with cholinoreceptors in rat submandibular gland-dependent stimulation of cyclooxygenase 2 (COX-2) mRNA expression and PGE(2) production. Our findings indicated that pSS IgG-stimulating M(3), M(4), and M(1) cholinoreceptors exerted an increase in COX-2 mRNA without affecting COX-1 mRNA expression and increased PGE(2) production. Inhibitors of phospholipase A(2), COX- s, L-type calcium channel currents, and Ca(2+)-ATPase from sarcoplasmic reticulum prevented the pSS IgG effect on PGE(2) production. An ionophore of calcium mimicked pSS IgG action, suggesting a crucial role of calcium homeostasis in the cholinoreceptor-stimulated increase in PGE(2) production. Moreover, the amounts of PGE(2) in saliva and in sera from persons with pSS were significantly higher than in pre- or post-menopausal women. These findings illustrate the importance of autoantibodies to cholinoreceptors in the generation of chronic inflammation of target tissues in SS.

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

Effects of celecoxib on voltage-gated calcium channel currents in rat pheochromocytoma (PC12) cells

Cyclooxygenase-2 (COX-2) plays crucial roles in the development and invasion of tumors. Celecoxib, a selective COX-2 inhibitor, has been shown to be chemopreventive against cancer. However, to date, the mechanisms of these effects remain unclear. In this study, we investigate the effects of celecoxib on voltage-gated calcium channel (VGCC) currents in undifferentiated pheochromocytoma (PC12) cells using whole-cell patch clamp. Our results showed that celecoxib, instead of rofecoxib or NS-398, another selective COX-2 inhibitor, reversibly inhibited the current density of VGCC in a concentration-dependent manner, but had no apparent effects on the cells treated with nifedipine (1 μM), an L-type calcium channel blocker. Upon pre-incubation of PC12 cells with omega-conotoxia GVIA (1 μM), an N-type calcium channel blocker, omega-agatoxin IVA (1 μM), a P/Q-type calcium channel blocker, or SNX-482 (1 μM), a R-type calcium channel blocker, celecoxib (1 μM) inhibited the currents by 36%, 28%, and 25%, respectively. Celecoxib up-shifted the current–voltage ( I– V), and hyperpolarizedly shifted the inactivation curve, but did not markedly affect the activation curve. Intracellular application of H89, a protein kinase A inhibitor, failed to affect the celecoxib's VGCC currents inhibition. Taken together, our present results suggested that celecoxib inhibited L-type calcium channels in PC12 cells via a COX-2 independent pathway, which might be responsible for its clinical effects including anti-tumor.

Effects of spermine on the relaxation response of rat detrusor smooth muscles

Endogenous polyamines are known to influence excitation-contraction coupling in smooth muscle. This study was designed to determine the effects of the polyamines spermine, spermidine, and putrescine on the contractile responses of rat detrusor smooth muscles. Under physiological conditions, isometric tension recordings were made of isolated bladder strips from excised rat bladder. The effects of spermine, spermidine, and putrescine (1 mM each) on the bladder contractions induced by various agents, i.e., acetylcholine, bethanechol, high-K, and tetraethylammonium (TEA) were measured. A conventional patch clamp technique was used in whole cell mode with single smooth muscle cells of rat bladder. Calcium channel currents were recorded to determine the effects of spermine on channel activities. Polyamines elicited a concentration-dependent relaxations on the contractile agents induced contractures. Spermine showed the most potent relaxation effect of the polyamines examined, and relaxed the contractions induced by the agents. Calcium channel activities were significantly reduced by adding 1 mM spermine to the bath. We concluded that spermine exerts a potent relaxant effect on rat bladder smooth muscle, and this effect appears to be mediated by calcium channel antagonism.

The ω-atracotoxins: Selective blockers of insect M-LVA and HVA calcium channels

The ω-atracotoxins (ω-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, ω-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the ω-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets . Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1 μM. To further characterize the target of the ω-ACTXs, voltage-clamp analysis using the whole-cell patch-clamp technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that ω-ACTX-Ar1a, and its homolog ω-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid–low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Ca v) currents. This block occurred in the absence of alterations in the voltage-dependence of Ca v channel activation, and was voltage-independent, suggesting that ω-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 μM ω-ACTX-Ar1a failed to significantly affect global K v channel currents. However, 1 μM ω-ACTX-Ar1a caused a modest 18% block of insect Na v channel currents, similar to the minor block of Na v channels reported for other insect Ca v channel blockers such as ω-agatoxin IVA. These findings validate both M-LVA and HVA Ca v channels as potential targets for insecticides.

Effect of ceramide on the contractility of pregnant rat uterus

Ceramide and other sphingolipid mediators have emerged as a novel class of lipid second messengers in cell signaling. We assessed the effect of C 2-ceramide (a membrane permeable analog of ceramide) on spontaneous and agonist-induced contractile responses of uterus, isolated from 19-day pregnant rats. Ceramide (3, 10 μM) moderately, but significantly inhibited the amplitude of spontaneous rhythmic contractions. However, a variable effect was seen on agonist-induced contractions. While 5-HT-induced contractions were markedly inhibited at 3 and 10 μM ceramide, oxytocin and carboprost (a PGF 2α analogue)-induced contractions were not affected by the sphingolipid. Ceramide (10 μM) also markedly inhibited CaCl 2-induced contractions elicited in K +-depolarized tissues. Further, in rabbit portal vein myocytes, which display robust L-type calcium channel current, ceramide inhibited the I Ba in a dose-dependent manner. Therefore, it is suggested that the inhibitory effect of ceramide on uterine contractility may involve a decrease in the influx of Ca 2+ through voltage-dependent L-type Ca 2+ channels, such that contractile responses that are primarily dependent on extracellular Ca 2+, like rhythmic and serotonin contractions, were inhibited by ceramide. Further study is required to establish the role of endogenous ceramide and other sphingolipids in regulating uterine tone during gestation and at term.

Effects of extracellular pH on neuronal calcium channel activation

Previous studies have shown that extracellular pH (pH o) alters gating and permeation properties of cardiac L- and T-type channels. However, a comprehensive study investigating the effects of pH o on all other voltage-gated calcium channels is lacking. Here, we report the effects of pH o on activation parameters slope factor (S), half-activation potential (V a), reversal potential (E rev), and maximum slope conductance (G max) of the nine known neuronal voltage-gated calcium channels transiently expressed in tsA-201 cells. In all cases, acidification of the extracellular bathing solution results in a depolarizing shift in the activation curve and reduction in peak current amplitudes. Relative to a physiological pH o of 7.25, statistically significant depolarizing shifts in V a were observed for all channels at pH o 7.00 except Ca v1.3 and 3.2, which showed significant shifts at pH o 6.75 and below. All channels displayed significant reductions in G max relative to pH o 7.25 at pH o 7.00 except Ca v1.2, 2.1, and 3.1 which required acidification to pH o 6.75. Upon acidification Ca v3 channels displayed the largest changes in V as and exhibited the largest reduction in G max compared with other channel subtypes. Taken together, these results suggest that significant modulation of calcium channel currents can occur with changes in pH o. Acidification of the external solution did not produce significant shifts in observed E revs or blockade of outward currents for any of the nine channel subtypes. Finally, we tested a simple Woodhull-type model of current block by assuming blockade of the pore by a single proton. In all cases, the amount of blockade observed could not be explained in these simple terms, suggesting that proton modulation is more complicated, involving more than one site or gating modification as has been previously described for cardiac L- and T-type channels.

Cellular and molecular determinants of altered Ca2+handling in the failing rabbit heart: primary defects in SR Ca2+uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.

Mechanism of Cav1.2 channel modulation by the amino terminus of cardiac β2‐subunits

L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor Gö-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or Gö-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.

Calcium channel subtypes – another layer of complexity to an already intricate story

Calcium channel subtypes – another layer of complexity to an already intricate story

IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells

We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0 ± 9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3–4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6 ± 2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1 ± 9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

Heterogeneous signaling pattern of PKC activity associated with the Ca v 1.2 calcium channel current

Heterogeneous signaling pattern of PKC activity associated with the Ca _v 1.2 calcium channel current

Effects of Polyamines on Contractility of Guinea-Pig Gastric Smooth Muscle

This study was designed to investigate the effects of polyamines on mechanical contraction and voltage-dependent calcium current (VDCC) of guinea-pig gastric smooth muscle. Mechanical contraction and calcium channel current (IBa) were recorded by isometric tension recording and whole-cell patch clamp technique. Spermine, spermidine and putrescine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner. Spermine (2 mM) reduced high K+ (50 mM)-induced contraction to 16±6.4% of the control (n=9), and significantly inhibited IBa in a reversible manner (p<0.05; IC50=0.8 mM). Pre- and post-treatment of tissue with spermine (2-5 mM, n=10) also inhibited acetylcholine (10 µM)-induced phasic contraction to 5±6.4% of the control. Inhibitory effect of spermine on IBa was observed at a wide range of test potentials of current/voltage (I/V) relationship (p<0.05), and steady-state activation of IBa was shifted to the right by spermine (p<0.05). Spermidine and putrescine (1 mM each) also inhibited IBa to 51±5.7% and 81±5.3% of the control, respectively. And putrescine (1 mM) inhibited IBa at whole tested potentials (p<0.05) without significant change of kinetics (p<0.05). Finally, 5 mM putrescine also inhibited high K+-induced contraction to 53±7.1% of the control (n=4). These findings suggest that polyamines inhibit contractions of guinea-pig gastric smooth muscle via inhibition of VDCC.

Comparison of effects of PKA catalytic subunit on Ih and calcium channel currents in rat dorsal root ganglion cells

We investigated whether PKA-induced phosphorylation was involved in regulation of hyperpolarization-activated current (I(h)) in rat dorsal root ganglion (DRG) cells. We examined the effect of the catalytic subunit of PKA (PKAc) on I(h) and confirmed an effect of PKAc on Ca(2+) channel currents carried by Ba(2+) (I(Ba)) in identical neurons as a positive control of PKA activity. After the start of recording, amplitudes of I(Ba) gradually decreased (rundown). An intracellular application of ATP reduced the rundown of I(Ba) and induced a depolarizing shift of I(h) activation. The former was partially reversed by PKI but the latter was not affected. An intracellular application of PKAc also prevented the rundown of I(Ba) and this effect was potentiated by okadaic acid (OA). The application of PKAc and OA in combination did not change the electrophysiological properties of I(h) although a potentiating effect on I(Ba) was observed in the same neurons. The application of 2-mM ATP in addition to PKAc and OA did not result in an additional potentiation of I(Ba), but shifted the activation curve of I(h) positively. These results suggested that PKA-induced phosphorylation was not involved in the modulatory mechanisms of I(h) in rat DRG neurons.

Protein kinase C contributes to enhanced calcium sensitivity in coronary arteries from hypercholesterolemic swine

Previous studies have reported enhanced KCl-mediated constriction despite reduced smooth muscle cell calcium channel current in coronary arteries from hypercholesterolemic swine. We tested the hypothesis that coronary arteries of hypercholesterolemic pigs are more sensitive to calcium due to an activation of protein kinase C (PKC). Male Yucatan miniature swine were randomly assigned to a control (CON) or high fat/high cholesterol (HC) diet for 23 wks and then killed. Vascular reactivity of rings isolated from the left anterior descending coronary artery was studied using standard isometric techniques. Rings from HC animals constricted with greater force per unit of intracellular calcium in response to KCl. Additionally, the response to increasing calcium concentrations was greater in permeabilized rings from HC compared with CON animals. Rings isolated from HC animals also exhibited enhanced maximal constriction to KCl. The broad-spectrum PKC inhibitor calphostin C (1 μM) attenuated this constriction to a greater extent in arteries from HC compared to CON animals; however, the conventional PKC isoform-selective inhibitor bisindolylmaleimide (1 μM) had no effect in either group. We conclude that the increase in calcium sensitivity of the coronary vasculature in HC animals is attributable at least in part to an increased contribution of the novel PKC isoforms. Support: AHA 0330252N