Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: biochemical characterization and autoradiographic localization

CRF receptors were characterized using radioligand binding and chemical affinity cross-linking techniques and localized using autoradiographic techniques in porcine, bovine and rat pituitaries. The binding of 125I-[Tyr0]-ovine CRF (125I-oCRF) to porcine anterior and neurointermediate lobe membranes was saturable and of high affinity with comparable KD values (200-600 pM) and receptor densities (100-200 fmoles/mg protein). The pharmacological rank order of potencies for various analogs and fragments of CRF in inhibiting 125I-oCRF binding in neurointermediate lobe was characteristic of the well-established CRF receptor in anterior pituitary. Furthermore, the binding of 125I-oCRF to both anterior and neurointermediate lobes of the pituitary was guanine nucleotide-sensitive. Affinity cross-linking studies revealed that the molecular weight of the CRF binding protein in rat intermediate lobe was identical to that in rat anterior lobe (Mr = 75,000). While the CRF binding protein in the anterior lobes of porcine and bovine pituitaries had identical molecular weights to CRF receptors in rat pituitary (Mr = 75,000), the molecular weight of the CRF binding protein in porcine and bovine intermediate lobe was slightly higher (Mr = 78,000). Pituitary autoradiograms from the three species showed specific binding sites for 125I-oCRF in anterior and intermediate lobes, with none being apparent in the posterior pituitary. The identification of CRF receptors in the intermediate lobe with comparable characteristics to those previously identified in the anterior pituitary substantiate further the physiological role of CRF in regulating intermediate lobe hormone secretion.     

Molecular and Pharmacological Characterization of GABAA Receptors in the Rat Pituitary

Abstract: Levels of mRNA for the major subunits of the GABA_A receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. α1, β1, β2, β3, and γ2s were found to be the predominant subunits in the anterior lobe, whereas α2, α3, β1, β3, γ2s, and γ1 were the predominant subunits expressed in the neurointermediate lobe. α5, α6, and δ subunits were not detectable. Hill and Scatchard analysis of [³H]muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 n M , respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [³H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 n M in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218 872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABA_A receptor subunits and receptor heterogeneity within individual cells in the pituitary.     

Alpha1 Adrenergic Receptors in the Porcine Pituitary Neurointermediate Lobe: Detection with [3H]Ketanserin

Abstract

[³H]Ketanserin, a serotonin receptor antagonist, labelled high affinity, saturable sites in homogenates of porcine neurointermediate lobe tissue. Cinanserin, a potent and selective serotonin receptor antagonist, inhibited the specific binding of 5 × 10^-10M [³H]ketanserin with a high affinity component representing 20% of the total binding. Prazosin, a potent and selective alpha₁ adrenergic antagonist, inhibited [³H]ketanserin binding with a high affinity component representing 60% of total binding. The prazosin-specific component was demonstrated to be distinct from the cinanserin-specific component. 10^-7M cinanserin was co-incubated with [³H]ketanserin to eliminate the serotonergic component of the binding and allow pharmacological characterization of the remaining prazosin-specific component. The prazosin-specific binding of [³H]ketanserin binding closely resembled the results of experiments using [³H]prazosin to label alpha₁ receptors in neurointermediate lobe tissue homogenates. Ketanserin was found to be sevenfold more potent in inhibiting [³H]prazosin binding to alpha₁ adrenergic receptors in the neurointermediate lobe tissue than in brain tissue. This observation explains why low concentrations of [³H]ketanserin can selectively label serotonin receptors in the brain but will label both adrenergic and serotonin receptors in the neurointermediate lobe.     

Dihydropyridine Binding Sites Regulate Calcium Influx Through Specific Voltage-Sensitive Calcium Channels in Cerebellar Granule Cells

In primary cultures of cerebellar granule cells, [3H]nitrendipine binds with high affinity to a single site (KD 1 nM and Bmax 20 fmol/mg protein). The 1,4-dihydropyridine (DHP) class of compounds such as nitrendipine, nifedipine, and BAY K 8644 displace [3H]nitrendipine binding at nanomolar concentrations. Verapamil partially inhibits whereas diltiazem slightly increases the [3H]nitrendipine binding. In these cells, the calcium influx that is induced by depolarization is very rapid and is blocked by micromolar concentrations of inorganic calcium blockers such as cadmium, cobalt, and manganese. The calcium influx resulting from cell depolarization is potentiated by BAY K 8644 and partially inhibited (approximately 40%) by nitrendipine and nifedipine. Other non-DHP voltage-sensitive calcium channel (VSCC) antagonists, such as verapamil and diltiazem, completely blocked the depolarization-induced calcium influx. This suggested that nitrendipine and nifedipine block only a certain population of VSCCs. In contrast, verapamil and diltiazem do not appear to be selective and block all of VSCCs. Perhaps some VSCCs can be allosterically modulated by the binding site for the DHPs, whereas verapamil and diltiazem may block completely the function of all VSCCs by occupying a site that differs from the DHP binding site.     

Identification of Neurotensin Receptors Associated with Calcium Channels and Prolactin Release in Rat Pituitary

Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 +/- 11 fmol/mg protein and a KD value of 1.4 +/- 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L-Phe-NT, and 3,000 nM for the pharmacologically inactive form D-Phe-NT. NT, up to a concentration of 1 microM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time- and dose-dependent. It appeared to be associated with neurotransmitter-operated calcium channels since: preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and NT lost its capacity to release PRL in the absence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]nitrendipine binding to adrenal capsular membranes

The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. We therefore studied the [3H]nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with KD = .26 +/- .04 nM and Bmax = 105 +/- 5.7 fmol/mg protein. Specific binding of [3H]nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine much greater than verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for [3H]nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production.     

High affinity binding sites for [3H]-nitrendipine in rabbit uterine smooth muscle

The binding properties of the high affinity binding site for [³H]-nitrendipine on rabbit uteri membranes were investigated. The specific component had a dissociation constant of 0.46±.07nM and a maximal binding of 175±11 pmol/mg. A variety of other calcium channel blockers inhibited the binding of [³H]nitrendipine with varying potencies. Flunarizine demonstrated a high potency (IC₅₀ = 0.30 uM) in inhibiting binding while verapamil and bepridil were less potent with IC₅₀'s of approximately 0.6–1.5 uM. Diltiazem did not displace nitrendipine even at very high concentrations. Verapamil displayed negative cooperative inhibition suggesting that a second site exists on uterine membranes for the binding of other calcium channel blockers.     

High affinity specific [3H] (+)PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat cerebral cortex and heart

The binding properties of the 1,4-dihydropyridine calcium channel antagonist, [³H](⁺)PN 200-110, were studied in rat cerebral cortical and cardiac homogenates (37°C, Krebs phosphate buffer). Specific binding of [³H](⁺)PN 200-110 was saturable, reversible, and of high affinity (K_d values are 35 and 64 pM for the cerebral cortex and heart, respectively). In parallel studies with [³H](⁺)PN 200-110, the dissociation constant of [³H]nitrendipine was 10–12 times higher. Substituted dihydropyridine calcium channel antagonists and agonists competitively inhibited specific [³H](⁺)PN 200-110 binding, but d-cis diltiazem enhanced and verapamil incompletely inhibited [³H](⁺)PN 200-110 binding in both the cerebral cortex and the heart. The effects of diltiazem and verapamil on [³H](⁺)PN 200-110 binding were due mainly to alterations in the dissociation constant (K_d), without alterations in the binding density (B_max). The new [³H](⁺)PN 200-110 receptor binding assay is remarkable for its low degree of nonspecific binding as compared to [³H]nitrendipine at physiological temperatures. [³H(⁺)PN 200-110 is a useful ligand for the further analysis of the dihydropyridine binding sites associated with calcium channels.     

Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein

The calcium channel blockers verapamil and diltiazem have been shown to reverse multidrug resistance, but the mechanism of action of these agents is still unknown. We measured [3H]verapamil, [3H]desmethoxyverapamil, [3H]diltiazem, and [3H]nitrendipine binding to membrane vesicles made from drug-sensitive (KB-3-1), multidrug-resistant (KB-C4 and KB-V1), and revertant (KB-V1-R2) cells. Membrane vesicles from KB-V1 cells bound 10-20-fold more [3H]verapamil and [3H]diltiazem and about 30-fold more [3H]desmethoxyverapamil than did vesicles from the parental KB-3-1 or revertant KB-V1-R2 cell lines. These drugs reverse the multidrug resistance phenotype by increasing accumulation of drugs in the resistant cells. No difference in binding of [3H]nitrendipine, which did not reverse drug resistance, was observed. The binding of vinblastine, desmethoxyverapamil, and diltiazem to KB-V1 vesicles was specific and saturable and was inhibited by desmethoxyverapamil and quinidine greater than vinblastine and diltiazem much greater than daunomycin. In addition, verapamil and diltiazem inhibited the vinblastine photoaffinity labeling of P170, the protein previously shown to be a marker of multidrug resistance.     

Effect of calcium channel modulators on isolated endothelial cells

Indirect evidence, using organic calcium channel modulators suggests that calcium channels exist in endothelial cells. Using freshly prepared and cultured bovine aortic endothelial cells, we have studied the effect of calcium channel modulators on Fura-2 fluorescence and have examined the binding of the dihydropyridine, (+)[3H]PN200-110. In both isolated primary and cultured cells, external calcium (0.5-2 mM) and bradykinin (10(-8) M) increased the intracellular calcium concentration. In cultured cells, the increase in calcium was not significantly attenuated by preincubation with nitrendipine (10(-8) M) or d-cis-diltiazem (10(-6) M). The calcium agonists (-)Bay k8644 and (+)202-791 had no effect on intracellular calcium concentration, but other agonists including ATP (10(-4) M) and thrombin (1.5 micrograms/ml) significantly increased the calcium concentration. Competition binding studies with (+)[3H]PN200-110 indicated specific binding of this ligand with a KD of 57 nM and a Bmax of 2.1 pmol/10(6) cells. While these data do not provide convincing evidence for the existence of calcium channels in cultured or fresh bovine aortic endothelial cells, explanations may yet reconcile our observations with the presence of calcium channels in these cells.     

Membrane-associated forms of peptidylglycine alpha-amidating monooxygenase activity in rat pituitary. Tissue specificity

Membrane-associated peptidylglycine alpha-amidating monooxygenase (PAM) activity was investigated in rat anterior and neurointermediate pituitary tissues and in pituitary AtT-20/D-16v and GH3 cell lines. A substantial fraction of total pituitary PAM activity was found to be membrane-associated. Triton X-100, N-octyl-beta-D-glucopyranoside, and Zwittergent were effective in solubilizing PAM activity from crude pituitary membranes. The distribution of enzyme activity between soluble and membrane-associated forms was tissue-specific. In the anterior pituitary lobe and pituitary cell lines, 40-60% of total PAM activity was membrane-associated while only 10% of the alpha-amidating activity in the neurointermediate lobe was membrane-associated. Soluble and membrane-associated forms of PAM shared nearly identical characteristics with respect to copper and ascorbate requirements, pH optima, and Km values. Upon subcellular fractionation of anterior and neurointermediate pituitary lobe homogenates on Percoll gradients, 12-18% of total PAM activity was found in the rough endoplasmic reticulum/Golgi fractions and 42-60% was localized to secretory granule fractions. For both tissues, membrane-associated PAM activity was enriched in the rough endoplasmic reticulum/Golgi pool, whereas most of the secretory granule-associated enzyme activity was soluble.     

Different localization of receptors for omega-conotoxin and nitrendipine in rat brain

The bindings of radioiodinated omega-conotoxin GVIA and [3H]-nitrendipine to subcellular fractions of rat brain were examined. The results indicated that omega-conotoxin binding site was mainly present in the mitochondrial fraction, whereas nitrendipine binding site was rich in the mitochondrial but also present in the post-mitochondrial fraction. Fractionation of the mitochondrial fraction on a sucrose density gradient centrifugation showed that the both binding sites were localized in the heavy synaptosomal fraction. These results strongly suggest that the N- and L-type voltage-sensitive calcium channels have different localizations.     

Altered transverse tubule dihydropyridine receptor binding in malignant hyperthermia

Transverse tubule (TT) membrane vesicles have been isolated from the skeletal muscle of normal and malignant hyperthermia-susceptible (MHS) pigs. MHS and normal TT did not differ in the distribution of the major proteins, cholesterol, or phospholipid content, (Na+ + K+)-ATPase activity, [3H]ouabain binding, Ca2+-ATPase activity, Mg2+-ATPase activity, or [3H]saxitoxin binding. Furthermore, in the presence of micromolar Ca2+, MHS and normal TT did not differ significantly in the KD values for either [3H]nitrendipine binding (2.7 +/- 0.6 and 3.3 +/- 0.5 nM, respectively) or (-)-[3H]desmethoxyverapamil ([3H]D888) binding (7.2 +/- 0.9 and 6.4 +/- 0.6 nM, respectively). However, in contrast to normal TT, MHS TT exhibited a significantly decreased Bmax for both [3H]nitrendipine binding (26.4 +/- 5.4 for MHS versus 40.6 +/- 3.7 pmol/mg protein for normal TT) and [3H]D888 binding (17.8 +/- 7.0 for MHS versus 37.4 +/- 5.9 pmol/mg protein for normal TT). At calcium concentrations greater than 0.1 mM, there was a greater inhibition of [3H]nitrendipine binding to normal than to MHS TT such that binding was now similar for both preparations. As with purified TT, [3H]nitrendipine binding to MHS muscle homogenates was significantly less than to normal muscle homogenates (109 +/- 20 versus 211 +/- 19 fmol/mg protein, for MHS and normal TT, respectively); this difference was not apparent when 100 mM CaCl2 was included in the binding medium. We conclude that the altered MHS TT dihydropyridine receptor properties may reflect an adaptation of the TT voltage sensing mechanism to the abnormal sarcoplasmic reticulum calcium release channel regulation in MHS muscle.     

Ion pathways in transverse tubules. Quantification of receptors in membranes isolated from frog and rabbit skeletal muscle

The presence of four cation pathways in membrane vesicles isolated from transverse tubules of frog and rabbit skeletal muscle was studied by measuring binding of specific blockers. Transverse tubules purified from frog muscle have a maximal binding capacity for [3H]nitrendipine (a marker for voltage-dependent calcium channels) of 130 pmol/mg of protein; this binding is strongly dependent on temperature and, at 37 degrees C, on the presence of diltiazem. Receptors for [3H]ethylenediamine tetrodotoxin (a marker for voltage-dependent sodium channels) and for 125I-labeled alpha-bungarotoxin (a marker for acetylcholine-mediated channels) showed maximal binding values of about 5 pmol/mg. The number of sodium-pumping sites in the isolated tubule vesicles, inferred from [3H]ouabain binding, was 215 pmol/mg. The high purity of this preparation makes feasible the use of these values as a criterion to judge the degree of purity of isolated preparations, and it allows investigation of transverse tubule contamination in other muscle membrane fractions.     

Increased Activation of L-Type Voltage-Dependent Calcium Channels Is Associated with Glycine Enhancement of N-Methyl-d-Aspartate-Stimulated Dopamine Release in Global Cerebral Ischemia/Reperfusion

Abstract: We investigated the relationships among N -methyl- d -aspartate, glycine, L-type voltage-dependent calcium channels, and [³H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [³H]PN200-110 ([³H]isradipine) to L-type voltage-dependent calcium channels, that open as a consequence of N -methyl- d -aspartate-induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage-sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N -methyl- d -aspartate-stimulated [³H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N -methyl- d -aspartate-stimulated [³H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N -methyl- d -aspartate-stimulated release in all treatment groups. The enhancement of N -methyl- d -aspartate-stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30-min reperfused animals. These data suggest that glycine may facilitate opening of the voltage-dependent calcium channels activated by N -methyl- d -aspartate and that this facilitation is blocked by the antagonist nitrendipine.     

Relationship of Dihydropyridine Binding Sites with Calcium-Dependent Neurotransmitter Release in Synaptosomes

In the present work, we have studied the effect of ruthenium red (RuR), La3+ and 4-aminopyridine (4-AP) on the specific binding of (+)-[3H]PN200-110 to synaptosomes, as well as the effect of nitrendipine, nifedipine, and BAY K 8644 on gamma-[3H]aminobutyric acid [( 3H]GABA) release induced by potassium depolarization and by 4-AP in synaptosomes. Scatchard plots indicated that neither RuR nor 4-AP modifies the KD and Bmax of [3H]PN200-110 specific binding, whereas La3+ decreased the Bmax by about 25%; when the effect of the drugs on the total binding of PN200-110 was studied, a similar inhibition by La3+ was found. The calcium antagonists, nitrendipine and nifedipine, did not affect at all the potassium-stimulated release of [3H]GABA nor its release induced by 4-AP. The calcium agonist BAY K 8644 failed to affect both the spontaneous and the potassium-stimulated GABA release. Our results suggest that the binding sites of dihydropyridines in presynaptic membranes are not related to the calcium channels involved in neurotransmitter release with which RuR, La3+, and 4-AP interact.     

Evidence for intragranular processing of pro-opiocortin in the mouse pituitary intermediate lobe

Mouse pituitary neurointermediate lobes were pulse-incubated in [3H] arginine or [3H] lysine for 10 min and then chase-incubated for periods 0 to 4h. The labeled peptides from the lobes were analysed by immunoprecipitation with specific antisera, and thereafter, by acid-urea polyacrylamide gel electrophoresis. Using this paradigm, the synthesis of a prohormone common to adrenocorticotropin (ACTH) and endorphin was detected in 10 min pulse labeled lobes. Following a chase period, processing of the prohormone to several forms of ACTH (mol. wt. 25000, 23000, and 13000), beta-lipotropin and beta-endorphin was observed. To determine the intracellular site of processing of the prohormone, subcellular fractionation studies of labeled lobes were carried out. Analysis of the fractions from the pulse-labeled lobes revealed that the newly synthesized labeled prohormone was primarily localized in a granule-enriched fraction. In lobes that were pulsed and then chase-incubated for 1 h, there was a decrease in the amount of prohormone and an appearance of processed products in the granule-enriched fraction. In another paradigm, where the secretory granule-fraction was isolated from pulse-labeled lobes and then incubated in vitro for 6 h at pH 5.5, processing of the endogenous labeled prohormone within the isolated granule fraction was observed. These data suggest, that in the mouse neurointermediate lobe, the ACTH/endorphin prohormone (pro-opiocortin) is rapidly packaged into secretory granules after synthesis and processed intragranularly.     

3H]nitrendipine receptors in skeletal muscle

The richest source of receptors for the organic calcium channel blocker [3H]nitrendipine in muscle is the transverse tubule membrane. The tubular membrane preparation binds [3H]nitrendipine with a high affinity and has a very high number of [3H]nitrendipine binding sites. For example, for the transverse tubule membrane preparation from rabbit muscle, the dissociation constant of the nitrendipine-receptor complex is 1.8 +/- 0.3 nM and the maximum binding capacity Bmax = 50 +/- 6 pmol/mg of protein. Similar results have been found with a membrane preparation from frog muscle. The dissociation constant found at equilibrium is near that determined from the ratio of rate constants for association (kappa 1) and dissociation (kappa-1). Binding of [3H] nitrendipine is pH-dependent and reveals the presence of an essential ionizable group with a pK of 5.4 on the nitrendipine receptor. The binding is destroyed by proteases showing that the receptor is a protein. Three different classes of Ca2+ channel blockers inhibit [3H]nitrendipine to its specific site. (i) The dihydropyridine analogs of nitrendipine which are competitive inhibitors of [3H]nitrendipine. These molecules form tight complexes with the nitrendipine receptor with dissociation constants between 1.4 and 4.0 nM. (ii) Other antiarrhythmic molecules like verapamil, amiodarone, bepridil, and F13004 which are noncompetitive inhibitors of [3H]nitrendipine binding with dissociation constants between 0.2 and 1 microM. (iii) Divalent cations like Ni2+, Co2+, Mn2+, or Ca2+ which are noncompetitive inhibitors of [3H]nitrendipine binding with the following rank order of potency: Ni+ (K0.5 = 1.8 mM) greater than Co2+ (K0.5 = 2.7 mM) greater than Mn2+ (K0.5 = 4.8 mM) greater than Ca2+ (K0.5 = 65 mM).     

Identification of ω-Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells

Binding of 125I-omega-conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N- and L-type Ca2+ channels in adrenal chromaffin cells. Saturable, high-affinity binding sites for 125I-omega-conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 +/- 170 pM and a Bmax of 26 +/- 11 pmol/g of protein. 125I-omega-Conotoxin binding sites had a KD of 215 +/- 56 pM and a Bmax of 105 +/- 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I-omega-Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by omega-conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+-evoked cytosolic Ca2+ transients measured by fura-2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, omega-conotoxin had no effect on Bay K 8644-evoked Ca2+ transients. Thus, the binding sites for omega-conotoxin and for nitrendipine appear to be different. The results confirm the presence of L-type Ca2+ channels and open the possibility of N-type Ca2+ channels as the omega-conotoxin binding sites in chromaffin cell membranes.     

Novel 1,4-Dihydropyridine (Bay K 8644) Facilitates Calcium-Dependent [3H]Noradrenaline Release from PC 12 Cells

The effects of the novel 1,4-dihydropyridine Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate] on the release of [3H]noradrenaline in cultured PC 12 cells were investigated. K+ in a concentration-dependent manner evoked 3H-transmitter release with an EC50 of 50-56 mM. Bay K 8644 at 30 nM potentiated the K+-evoked [3H]noradrenaline release; however, in the absence of calcium neither K+ evoked nor Bay K 8644 enhanced [3H]noradrenaline release. At a K+ concentration of 25 mM, Bay K 8644 stimulated [3H]noradrenaline release fivefold, with an EC50 of 10 nM, and 100 nM of the calcium channel blocker nitrendipine shifted the concentration response curve of Bay K 8644 to the right in an apparently competitive fashion. Nitrendipine blocked the Bay K 8644-potentiated release with an EC50 of 700 nM in the presence of 500 nM Bay K 8644. [3H]Nitrendipine bound to a saturable population of binding sites on PC 12 cell membranes with a Bmax of 180 fmol X mg-1 of membrane protein and a KD of 0.9 nM. Bay K 8644 inhibited [3H]nitrendipine binding with a Ki of 16 nM. It is concluded that Bay K 8644 binds to, and stabilizes, the open state of calcium channels and thus acts as a "calcium agonist" to mediate calcium-dependent cellular events such as catecholamine release from PC 12 cells.