Dihydropyridine-sensitive Calcium Channels Research Articles

Postnatal laminar development of cholinergic receptors, protein kinase C and dihydropyridine-sensitive calcium antagonist binding in rat visual cortex. Effect of visual deprivation

The postnatal ontogeny of 3H-pirenzepine and 3H-oxotremorine-M binding to M1- and M2-muscarinic acetylcholine receptors, respectively, as well as 3H-nicotine binding to neuronal nicotinic acetylcholine receptors, 3H-phorbol-12,13-dibutyrate binding to protein kinase C and 3H-PN200-110 binding to dihydropyridine-sensitive calcium channels was studied in individual layers of the visual cortex in both normally raised and monocularly deprived rats (one eyelid sutured at the age of 11 days) using quantitative receptor autoradiography. Postnatal ontogeny of M1-muscarinic receptors is similar in each visual cortical layer reaching the highest receptor density at the age of 15 days, whereas M2-muscarinic binding sites increase gradually from day 7 up to day 34. Highest 3H-nicotine binding is reached in all visual cortical layers at postnatal day 15 followed by a considerable decrease in binding sites until day 25. Phorbol ester binding rises considerably from birth until the age of 15 days reaching nearly the adult value in the upper layers, whereas in layers V and VI a marked decrease in binding levels until adulthood can be observed. The developmental course of 3H-PN200-110 binding sites is similar in all visual cortical layers and exhibits a moderate rise in binding sites between postnatal days 7 and 15. Monocular deprivation results in permanent changes in the developmental profiles of phorbol ester as well as calcium antagonist binding sites, whereas the alterations in muscarinic and nicotinic cholinergic receptors folloing monocular deprivation are only of transient nature. The data presented suggest that acetylcholine plays a modulatory role during a certain period of early postnatal maturation of the visual cortex by affecting both cholinergic receptors and associated second messenger cascades.

Evidence for a noradrenergic innervation to alpha 1A-adrenoceptors in rat kidney.

1. Experiments were undertaken to characterize the alpha 1-adrenoceptor subtype mediating vasoconstrictor responses to periarterial noradrenergic nerve stimulation (PNS) in the isolated perfused kidney of the rat. 2. Vasoconstrictor responses to nerve stimulation were inhibited by prazosin (10 nM), 5-methyl-urapidil (30 nM), and nitrendipine (1 microM) but were resistant to inhibition by chloroethylclonidine (100 microM). 3. 5-Methyl-urapidil (30 nM), chloroethylclonidine (100 microM) and nitrendipine (1 microM) did not inhibit the neuronal release of tritium from nerves loaded with [3H]-noradrenaline. 4. The results suggest that renovascular alpha 1A-adrenoceptors receive a noradrenergic innervation and that the innervated receptors are coupled to dihydropyridine-sensitive calcium channels.

Identification of two distinct proteins that are immunologically related to the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel.

The alpha 1 subunit of the dihydropyridine-sensitive calcium channel is a protein which is critical for excitation-contraction coupling and L-type calcium current in skeletal muscle. Using antibodies generated against peptides from three regions of the deduced amino acid sequence of the alpha 1 subunit, we have identified two distinct proteins in rabbit skeletal muscle. Both proteins appeared to be recognized by antibodies against the amino (N) terminus of the alpha 1 subunit sequence. One protein was also recognized by antibodies against an internal (I) region of the predicted sequence but not by antibodies against the carboxyl (C) terminus. In contrast, the other protein was recognized by antibodies against the carboxyl terminus but not by the antibodies against the internal region. We have designated these proteins pNI and pNC based on their patterns of antibody recognition. No protein was detected which was recognized by all three antibodies. pNI is the protein commonly identified as the alpha 1 subunit of the dihydropyridine-sensitive calcium channel. Of note is that pNI, which apparently lacks sequences from the predicted carboxyl tail, is the protein present in preparations which we have previously demonstrated contain dihydropyridine-sensitive calcium channel activity. pNC is herein identified as a skeletal muscle protein that is immunologically related to the alpha 1 subunit of the dihydropyridine-sensitive calcium channel. Its function is unknown. In addition to their distinct patterns of antibody recognition, pNI and pNC were also distinguishable by several other properties. pNC migrated as a protein of approximately 160 kDa in 5% sodium dodecyl sulfate-polyacrylamide gels versus approximately 165 kDa for pNI. pNI was enriched in transverse tubule membranes, whereas pNC was found to be enriched in triad and junctional sarcoplasmic reticulum membrane fractions and was not found in transverse tubule membranes. Under conditions in which pNI bound to wheat germ agglutinin-Sepharose, pNC did not bind. The results demonstrate that there are two proteins in skeletal muscle which are immunologically related to the alpha 1 subunit of the dihydropyridine-sensitive calcium channel but which are distinguishable by several biochemical and immunological characteristics.

On the mechanism of parathyroid hormone stimulation of calcium uptake by mouse distal convoluted tubule cells.

PTH stimulates transcellular Ca2+ absorption in renal distal convoluted tubules. The effect of PTH on membrane voltage, the ionic basis of the change in voltage, and the relations between voltage and calcium entry were determined on immortalized mouse distal convoluted tubule cells. PTH (10(-8) M) significantly increased 45Ca2+ uptake from basal levels of 2.81 +/- 0.16 to 3.88 +/- 0.19 nmol min-1 mg protein-1. PTH-induced 45Ca2+ uptake was abolished by the dihydropyridine antagonist, nifedipine (10(-5) M). PTH did not affect 22Na+ uptake. Intracellular calcium activity ([Ca2+]i) was measured in cells loaded with fura-2. Control [Ca2+]i averaged 112 +/- 21 nM. PTH increased [Ca2+]i over the range of 10(-11) to 10(-7) M. Maximal stimulation to 326 +/- 31 nM was achieved at 10(-8) M PTH. Resting membrane voltage measured with the potential sensitive dye DiO6(3) averaged -71 +/- 2 mV. PTH hyperpolarized cells by 19 +/- 4 mV. The chloride-channel blocker NPPB prevented PTH-induced hyperpolarization. PTH decreased and NPPB increased intracellular chloride, measured with the fluorescent dye SPQ. Chloride permeability was estimated by measuring the rate of 125I- efflux. PTH increased 125I- efflux and this effect was blocked by NPPB. Clamping voltage with K+/valinomycin; depolarizing membrane voltage by reducing extracellular chloride; or addition of NPPB prevented PTH-induced calcium uptake. In conclusion, PTH increases chloride conductance in distal convoluted tubule cells leading to decreased intracellular chloride activity, membrane hyperpolarization, and increased calcium entry through dihydropyridine-sensitive calcium channels.

CCKB receptor stimulation mediates [Ca2+]i increase but no PKC activation in Jurkat T-cells.

We have investigated the effect of cholecystokinin-octapeptide (CCK-8) on [Ca2+]i and protein kinase C (PKC) activity in Jurkat T-cells. CCK-8 produced a transient [Ca2+]i increase in the presence of extracellular Ca2+. While CCKB receptor antagonist L-365,260 abolished the elevation of [Ca2+]i, CCKA receptor antagonist L-364,718 was without effect. Moreover, the dihydropyridine calcium channel blocker nitrendipine was shown to block the observed calcium response. Results suggest that the calcium effect is caused by an interaction of CCK-8 with CCKB binding sites and an influx of external Ca2+ via dihydropyridine sensitive calcium channels might serve as a source for the increased [Ca2+]i. Because CCK-8 induced no PKC activation CCKB receptor mediated rise of intracellular calcium seems not to include activation of phospholipase C.

The gene for the dihydropyridine-sensitive calcium channel α2 subunit (CCHL2A) maps to the proximal region of mouse chromosome 5

A ratbrain cDNA probe for the gene encoding the α2 subunit of the dihydropyridine-sensitive L-type calcium channel was used as a hybridization probe for the Southern blot analysis of Chinese hamster × mouse somatic cell hybrids and the progeny of an intersubspecies backcross. This gene, termed Cchl2a, was mapped near the centromeric end of the Chromosome 5 linkage group with gene order: centromere- Pgy-1-Cchl2a-Il-6-Pgm-1.

Specific phosphorylation of a COOH-terminal site on the full-length form of the alpha 1 subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase.

The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.

Modulation of dihydropyridine-sensitive gastric mucosal calcium channels by GM 1-ganglioside

1. 1. A dihydropyridine-sensitive calcium channel complex was solubilized from gastric mucosal cell membranes and purified by affinity chromatography on wheat germ agglutinin. 2. 2. The calcium channel complex labeled with [ 3H]PN200-110, when reconstituted into phosphatidylcholine vesicles, exhibited active 45Ca 2+ uptake into intravesicular space as evidenced by La 3+ displacement and osmolarity studies. The channel complex responded in a dose-dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 μM exerted maximal inhibitory effect of 66% in 45Ca 2+ uptake. 3. 3. The uptake of 45Ca 2+ into vesicle-reconstituted gastric mucosal calcium channel complex was inhibited by GM 1-ganglioside. Maximum inhibitory effect was achieved at 10–15 nM gm 1, at which point a 74% decrease in 45Ca 2+ uptake occurred. Furthermore, gm 1 also inhibited dihydropyridine binding to gastric mucosal membranes, indicating the extracellular orientation of calcium channel domains for gm 1. 4. 4. The ability of GM 1 to modulate the intracellular calcium levels may be an important feature in gastric mucosal protection by this ganglioside.

Calcitonin gene-related peptide in noradrenergic transmission in rat hypothalamus.

In the present study, we examined the regulatory mechanisms of calcitonin gene-related peptide on norepinephrine release in rat hypothalamus. Calcitonin gene-related peptide inhibited the stimulation-evoked norepinephrine release from hypothalamic slices of Sprague-Dawley rats in a dose-dependent manner, although the peptide did not affect basal release of norepinephrine. The blockade of the alpha 2-adrenergic receptors by RX 781094 failed to modulate the inhibitory effects of calcitonin gene-related peptide on norepinephrine release. Pretreatment of slices with islet activating protein, a toxin that interferes with the coupling of the inhibitory receptors to adenylate cyclase, did not affect the suppression of norepinephrine release by calcitonin gene-related peptide. However, Bay K 8644, a dihydropyridine-sensitive calcium channel agonist, significantly reversed the inhibitory effects of calcitonin gene-related peptide on norepinephrine release. These results show that calcitonin gene-related peptide might inhibit norepinephrine release in rat hypothalamus, partially mediated by interactions with dihydropyridine-sensitive Ca2+ channels but not by interactions with presynaptic alpha 2-adrenergic receptors and inhibitory guanosine triphosphate binding proteins. Furthermore, the finding suggests the possible involvement of calcitonin gene-related peptide in the regulation of sympathetic nervous activity in the central nervous system.

A two-motif isoform of the major calcium channel subunit in skeletal muscle

Evidence is presented that two isoforms of the voltage-dependent, dihydropyridine-sensitive calcium channel α1, subunit are present in newborn and adult skeletal muscle and that expression of these isoforms is developmentally regulated. A voltage-dependent calcium channel α1, cDNA from newborn muscle was cloned and found to be identical to that published from the adult, except that it was 2 kb shorter owing to an internal deletion. Nucleotide sequences, Northern blots, reverse-transcriptase PCR experiments, and sequencing of the PCR product confirmed that a segment corresponding to the inner two repeats of the structural prototype four homologous motifs is missing from the immature isoform. Immunological studies using antisera raised against synthetic peptides that correspond to sequences in the two isoforms show that the abbreviated transcript is predominant in newborn muscle, whereas the four-repeat isoform is the major species in the adult.

Expression of dihydropyridine-sensitive brain calcium channels in the rat central nervous system

We have localized dihydropyridine (DHP)-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel α1 subunit (DHPR-B) is prominently localized in neuronal cells in the olfactory bulb, dentate gyrus, hippocampus, arcuate nucleus, paraventricular nucleus, ventromedial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pineal glands. DHP-sensitive calcium channel α1 subunit distribution has also been examined immunohistochemically with polyclonal antibodies raised against synthetic peptides specific for the DHPR-B α1 subunit protein. The results from immunohistochemistry were in good agreement with those from in situ hybridization. Thus, regional distribution and localization of DHPR-B mRNA and α1 subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium channel may play an important role in excitation-secretion coupling functions in the neuroendocrine system.

Calcium signaling and secretory responses in agonist-stimulated pituitary gonadotrophs

In cultured pituatary gonadotrophs, gonadotropin-releasing hormone (GnRH) caused dose-dependent and biphasic increases in cytoplasmic calcium concentration ([Ca 2+] i) and LH release. Both extra- and intracellular calcium pools participate in GnRH-induced elevation of [Ca 2+] i and LH secretion. The spike phase of the [Ca 2+] i response represents the primary signal derived predominantly from the rapid mobilization of intracellular Ca 2+. In contrast, the prolonged phase of the Ca 2+ signal depends exclusively on Ca 2+ entry from the extracellular pool. The influx of Ca 2+ occurs partially through dihydropyridine-sensitive calcium channels. Both [Ca 2+] i and LH responses to increasing concentrations of GnRH occur over very similar time scales, suggesting that increasing degrees of receptor occupancy are transduced into amplitude-modulated Ca 2+ responses, which in turn activate exocytosis in a linear manner. However, several lines of evidence indicated the complexity over the relationship between Ca 2+ signaling and LH exocytosis. In contrast to [Ca 2+] i measurements in cell suspension, single cell Ca 2+ measurements revealed the existence of a more complicated pattern of Ca 2+ response to GnRH, with a biphasic response to high agonist doses and prominent oscillatory responses to lower GnRH concentrations, with a log-linear correlation between GnRH dose and the frequency of Ca 2+ spiking. In addition, analysis of the magnitudes of the magnitudes of the [Ca 2+] i and LH responses of gonadotrophs to a wide range of GnRH concentrations in the presence and absence of extracellular Ca 2+, and to K + and phorbol ester stimulation, showed non-linearity between these parameters with amplification of [Ca 2+] i-mediated exocytosis. Studies on cell depleted of protein kinase C under conditions that did not change the LH pool suggested the participation of protein kinase C in this amplication, especially during the plateau phase of the secretory response to GnRH.

Modulation of dihydropyridine-sensitive calcium channels in heart cells by fish oil fatty acids.

The highly unsaturated n-3 fatty acids from fish oils, eicosapentaenoic acid [EPA; C20:5 (n-3)] and docosahexanoic acid [DHA; C22:6 (n-3)], prevent the toxicity of high concentrations of the cardiac glycoside ouabain to isolated neonatal rat cardiac myocytes. Arachidonic acid [C20:4 (n-6)] lacks such protective action. The protective effect of the n-3 fatty acids is associated with their ability to prevent high levels of cytosolic free calcium from occurring in response to the ouabain. This in turn results, at least in part, from a 30% reduction in calcium influx rate induced by the n-3 fatty acids. This protective effect is simulated by nitrendipine, a dihydropyridine inhibitor of the L-type calcium channels in cardiac myocytes. Nitrendipine (0.1 mM) alone, however, inhibits myocyte contractility, as do verapamil (10 microM) and diltiazem (1.0 microM). EPA or DHA (5 microM) blocks the inhibitory effects of nitrendipine but not those of verapamil or diltiazem. Bay K8644, a known dihydropyridine agonist of L-type calcium channels, produces a ouabain-like effect that is also prevented by EPA or DHA. Specific binding of [3H]nitrendipine to intact myocytes is noncompetitively inhibited by EPA or DHA in a manner that reduces the number of high- and low-affinity binding sites (Bmax) and increases their affinities. The fish oil fatty acids prevent calcium overload from ouabain and Bay K8644. They also prevent a calcium-depleted state in the myocytes caused by the L-type calcium channel blocker nitrendipine. The protective effects of the n-3 fatty acids appear to result from their modulatory effects on nitrendipine-sensitive L-type calcium channels.

Erythromycin stimulates ileal motility by activation of dihydropyridine-sensitive calcium channels

Erythromycin, a macrolide antibiotic, is a potent stimulant of small bowel motor activity (MA) which may motility either via the peptide motilin receptor or neural mechanisms. We hypothesized that erythromycin stimulates directly stimulates smooth muscle cells by a calcium-mediated event. Thus, we evaluated the effect of neuronal blockade with tetrodotoxin, muscarinic blockade with atropine, and opiate blockade with naloxone on erythromycin-stimulated MA in isolated perfused segments of rabbit terminal ileum. We also tested the effect of nonspecific calcium channel blockade (verapamil and cadmiun) and specific blockade (dihydroxypyridine and nichol) on erythromycin-stimulated MA. MA was measured with a multichannel continuous perfusion manometry catheter. Erythromycin caused a concentration-dependent increase in MA (ED 100 5 × 10 −4 M). Tetrodotoxin, atropine, and naloxone did not effect erythromycin-stimulated MA ( P > 0.05). Both verapamil (10 −7 M) and cadmium (10 −2-10 −4 M) inhibited erythromycin-stimulated MA. Selective blockade of “1” type calcium channels using dihydropyridine (10 −6 M) and “t” channels with nickel (10 −2-10 −4 M) both reversed erythromycin-stimulated MA. Since the isolated segments of terminal ileum were free of exogenous humoral and neural effects, these studies indicated that erythromycin directly stimulated MA in the terminal ileum. Furthermore, since tetrodotoxin, atropine, and naloxone did not inhibit this increase in MA, erythromycin acted by a mechanism which was independent of the intrinsic nervous and opiate systems. In conclusion, these data are consistent with the model that erythromycin stimulates ileal motility by a mechanism involving activation of dihydroxypyridine and nickel-sensitive calcium channels.

L-type calcium channels, potassium channels, and novel nonspecific cation channels in a clonal muscle cell line derived from embryonic rat ventricle.

We have characterized the membrane currents in the H9c2 clonal muscle cell line derived from embryonic rat ventricle. These cells, established by selective serial passage and clonal proliferation, have been found by Hescheler and coworkers to express dihydropyridine-sensitive calcium channels that respond to beta-adrenergic stimulation. We have investigated the macroscopic and elementary currents in these cells by using standard patch-clamp methods. In cells that are kept confluent for 3-4 weeks, we have confirmed the expression of L-type calcium channels and additionally establish that the unitary conductance of many, but not all, of these channels (25 pS in 70 mM barium) is equal to that of cardiac rather than skeletal muscle. When the cells are proliferating rapidly, calcium channels are sparse or absent, but at least two distinct potassium channels and a nonspecific cation channel are observed. The nonspecific channel exhibits a conductance of 30 pS in physiological saline and conducts sodium, potassium, and calcium with nearly equal efficacy. Several unusual properties distinguish this nonspecific channel from others described previously. Gating is voltage dependent, with slow activation and marked increases in open probability at positive potentials. Unlike voltage, activation and marked increases in open probability at positive potentials. Unlike voltage, changes in [Ca2+] or in membrane stretch do not noticeably influence activity. In conclusion, our work and that of Hescheler et al indicate that H9c2 cells are potentially valuable surrogates for the investigation of ion channel regulation and muscular gene expression.

Cloning and tissue-specific expression of the brain calcium channel β-subunit

A cDNA clone encoding a protein with high homology to the β-subunit of the rabbit skeletal muscle dihydropyridine-sensitive calcium channel was isolated from a rat brain cDNA library. This rat brain β-subunit cDNA hybridizes to a 3.4 kb message that is expressed in high levels in the cerebral hemispheres and hippocampus but is significantly reduced in cerebellum. The open reading frame encodes 597 amino acids with a predicted mass of 65 679 Da which is 82% homologous with the skeletal muscle β-subunit. The brain cDNA encodes a unique 153 amino acid C-terminus and predicts the absence of a muscle-specific 50 amino acid internal segment. It also encodes numerous consensus phosphorylation sites suggesting a role in calcium channel regulation. The corresponding human β-subunit gene was localized to chromosome 17. Hence the encoded brain β-Subunit. which has a primary structure highly similar to its isoform in skeletal muscle, may have a comparable role as an integral regulatory component of a neuronal calcium channel.

A monoclonal antibody to the beta subunit of the skeletal muscle dihydropyridine receptor immunoprecipitates the brain omega-conotoxin GVIA receptor.

Antibodies against the subunits of the dihydropyridine-sensitive L-type calcium channel of skeletal muscle were tested for their ability to immunoprecipitate the high affinity (Kd = 0.13 nM) 125I-omega-conotoxin GVIA receptor from rabbit brain membranes. Monoclonal antibody VD2(1) against the beta subunit of the dihydropyridine receptor from skeletal muscle specifically immunoprecipitated up to 86% of the 125I-omega-conotoxin receptor solubilized from brain membranes whereas specific antibodies against the alpha 1, alpha 2, and gamma subunits did not precipitate the brain receptor. Purified skeletal muscle dihydropyridine receptor inhibited the immunoprecipitation of the brain omega-conotoxin receptor by monoclonal antibody VD2(1). The dihydropyridine receptor from rabbit brain membranes was also precipitated by monoclonal antibody VD2(1). However, neither the neuronal ryanodine receptor nor the sodium channel was precipitated by monoclonal antibody VD2(1). The omega-conotoxin receptor immunoprecipitated by monoclonal antibody VD2(1) showed high affinity 125I-omega-conotoxin binding, which was inhibited by unlabeled omega-contoxin and by CaCl2 but not by nitrendipine or by diltiazem. An antibody against the beta subunit of the skeletal muscle dihydropyridine receptor stained 58- and 78-kDa proteins on immunoblot of the omega-conotoxin receptor, partially purified through heparin-agarose chromatography and VD2(1)-Sepharose chromatography. These results suggest that the brain omega-conotoxin-sensitive calcium channel contains a component homologous to the beta subunit of the dihydropyridine-sensitive calcium channel of skeletal muscle and brain.

Dihydropyridine-sensitive calcium channels from skeletal muscle. I. Roles of subunits in channel activity.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are hetero-oligomeric proteins. Little is known about the functional roles of the various subunits, except that the alpha 1 subunit is the essential channel unit. We have reconstituted both partially purified holomeric channels and the separated subunits into liposomes and measured their properties using an assay based on the Ca2+ indicator dye fluo-3. The holomeric channels exhibited Ca2+ influx that was sensitive to membrane potential achieved by the addition of valinomycin in the presence of a K+ gradient. Dissipation of the K+ gradient resulted in the loss of the valinomycin-sensitive Ca2+ flux. In addition, the reconstituted channels were: 1) activated by the dihydropyridine Ca2+ channel activator Bay K 8644 in a dose-dependent manner with a Kd of 20 nM; 2) inhibited by various types of Ca2+ channel inhibitors including the dihydropyridine (+)-PN 200-110, the phenylalkylamine verapamil, and the benzothiazepine d-cis-diltiazem; and 3) modulated in a stereoselective manner by the enantiomers of the dihydropyridine S-202-791. The purified channels used in this work possessed an alpha 1 subunit of 165 kDa and did not appear to contain a larger alpha 1 subunit of approximately 210 kDa, suggesting that channel activity with properties similar to those observed in intact cells can be supported with an alpha 1 subunit of 165 kDa. Reconstituted channels that were 85% depleted in the alpha 2/delta subunits showed a significant decrease in the initial rate of Ca2+ influx induced by valinomycin, but retained responsiveness to Bay K 8644 and (+)-PN 200-110. When the separated alpha 2 and delta subunits were added back to the alpha 1 subunit-containing preparation, the channels exhibited their normal rate of Ca2+ influx. These results demonstrated that the dihydropyridine-sensitive Ca2+ channels from skeletal muscle require the presence of the alpha 2.gamma complex in stoichiometric amounts to exhibit full activity.

Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.

Pressor actions of arginine vasopressin in pithed Sprague-Dawley, Wistar-Kyoto and spontaneously hypertensive rats before and after treatment with nifedipine or pertussis toxin.

The pressor actions of arginine vasopressin (AVP) were examined in pithed Sprague-Dawley and Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Prior to pithing, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded via an intra-arterial catheter from sodium pentobarbital anaesthetized rats. SBP and DBP recorded from SHR were significantly greater than those from Sprague-Dawley and WKY rats. However, after pithing, there were no significant differences between DBP among the various strains. Pertussis toxin pretreatment significantly reduced the prepithing SBP and DBP of the SHR but not Sprague-Dawley or WKY rats. Administration of nifedipine significantly reduced DBP of pithed rats. The dose-diastolic pressure response curves obtained from infusion of AVP in Sprague-Dawley and WKY rats were not significantly different from one another, but the maximal vasopressor responses to AVP in pithed SHR were enhanced. Administration of nifedipine to Sprague-Dawley and WKY rats did not affect the dose-response curve to AVP, but nifedipine administration in SHR led to a significant inhibition of the pressor responses to AVP. Furthermore, pertussis toxin pretreatment of rats significantly reduced a component of the AVP pressor effect in SHR but not Sprague-Dawley or WKY rats. We speculate that, in SHR, vasopressin receptors are coupled to a pertussis toxin-sensitive G protein that, in turn, may couple to a dihydropyridine-sensitive calcium channel and also to a pertussis-insensitive G protein that is probably coupled to the phospholipase C/intracellular calcium release process. A component of the elevated blood pressure in SHR is also regulated by a pertussis toxin-sensitive process.(ABSTRACT TRUNCATED AT 250 WORDS)