Neurotensin elevates cytosolic calcium in small cell lung cancer cells

The ability of neurotensin (NT) to elevate cytosolic Ca²⁺ in small cell lung cancer (SCLC) cells was investigated using the fluorescent Ca²⁺ indicator Fura 2-AM. Using SCLC cell line NCI-H345, NT elevated cytosolic Ca²⁺ levels in a concentration-dependent manner. Using a 10 nM dose, NT and C-terminal fragments such as NT(8–13) but not N-terminal fragments such as NT(1–8) elevated the cytosolic Ca²⁺ levels. Because EGTA (5 mM) did not affect the NT response, NT may cause release of Ca²⁺ from intracellular stores. These data indicate that SCLC NT receptors may use Ca²⁺ as a second messenger.     

Tumour-promoting phorbol esters inhibit agonist-induced phosphatidate formation and Ca flux in human platelets

Tumour-promoting phorbol esters (phorbol-12-myristate-13-acetate, PMA; phorbol-12,13-dibutyrate, PDBu) but not 4β-phorbol, activate protein kinase C. Using human platelets pre-labelled with quin2 or ³²PO₄ we examined the effects of these compounds on human platelet cytosolic free Ca²⁺ ([Ca²⁺]_j) and on [³²]phosphatidic acid ([³²P]PtdOH). PMA and PDBu, but not 4β-phorbol inhibited thrombin-, PAF- and vasopressin-induced elevation of [Ca²⁺], and [²⁺P]PtdOH formation. It is suggested that protein kinase C may act to terminate the transduction processes that link receptor occupancy to cellular activation.     

Hindlimb paralytic effects of arginine vasopressin and related peptides following spinal subarachnoid injection in the rat

Intrathecal (IT) injection of arginine vasopressin (AVP) in rats caused a transient (<30 min), dose-related paralysis of the hindlimbs, loss of hindlimb and tail nociceptive responsiveness, and increased mean arterial pressure. Motor dysfunction was produced with comparable potency by lysine vasopressin (LVP) and arginine vasotocin (AVT); oxytocin (OXY) was approximately 1000 times less potent. Paralysis induced by these peptides was selectively blocked following IT pretreatment with 0.5 nmoles of the vasopressin V₁ receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propioinic acid), 2-(O-methyl)tyrosine] Arg⁸-vasopressin (d(CH₂)₅[Tyr(Me²)]AVP). Pressor and antinociceptive responses to AVP were also blocked by this compound. However, at higher doses (2–5 nmoles, IT), d(CH₂)₅[Tyr(Me²)]AVP produced hindlimb paralysis, antinociception, and pressor responses by itself. In contrast to the fiber degeneration, cell loss, and necrosis found in lumbosacral cords of rats persistently paralyzed by other peptides (dynorphin A, somatostatin, and ICI 174864), neuropathological changes were not evident in spinal cords of rats transiently paralyzed by IT AVP. These results indicate that AVP-related peptides affected diverse spinal cord functions through interactions with a V₁-like receptor. The similar pattern of cardiovascular and antinociceptive responses to other peptides (dynorphin A, somatostatin, and ICI 174864), which also caused hindlimb paralysis, suggests that the former responses may actually reflect the nonselective consequences of a peptide-induced disruption of spinal cord function, rather than specific shared pharmacological effects.     

Characterization of Acetylcholine-induced Increases in Cytosolic Free Calcium Concentration in Individual Rat Pancreatic β-cells

The intracellular free Ca²⁺ ion concentration ([Ca²⁺]i) was measured using fura-2 microspec-trofluorimetry in individual rat pancreatic β-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca²⁺]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca²⁺ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca²⁺]i spikes of increasing amplitude and duration in some, but not all of the β-cells. In addition, the β-cells demonstrated variable sensitivity to ACh. The increases in [Ca²⁺]i were rapid, transient and were blocked by atropine at 10^-6M. A brief exposure to 50 mM K⁺ resulted in a transient increase in [Ca²⁺]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10^-4M or 10^-3M) induced [Ca²⁺]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca²⁺ free medium, the source of the increase in [Ca²⁺]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on [Ca²⁺]i. These data strongly suggest that ACh exerted a direct action on [Ca²⁺]i in normal rat pancreatic β-cells and support a role for Ca²⁺ as a second messenger in the action of ACh.     

Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth

Human small cell lung cancer (SCLC) produces and secretes BN/GRP (bombesin/gastrin releasing peptide). Because BN stimulates the growth of SCLC cells and these cells have receptors for BN-like peptides, it is important to define agents which disrupt this self-promoting autocrine growth cycle. Here, substance P analogues were evaluated as BN receptor antagonists using SCLC cell lines. (D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹) substance P [(APTTL)SP] was one of the more potent analogues tested in inhibiting BN-like peptide receptor binding with an IC₅₀ value of 1 μM. Micromolar concentrations of (APTTL)SP antagonized BN receptor mediated elevation of cytosolic Ca²⁺ levels and decreased the colony formation in soft agarose. These data suggest that SP analogues function as SCLC BN receptor antagonists and may be useful in disrupting the autocrine growth function of BN-like peptides.     

Hypoosmotic shock activates Ca channels in isolated nerve terminals

Influence of hypotonic swelling on Ca²⁺ (⁴⁵Ca²⁺) uptake in rat brain synaptosomes was studied. A decrease in medium osmolality from 310 to 260-180 mOsm led to a progressive stimulation of ⁴⁵Ca²⁺ accumulation. The effect was blocked by verapamil (IC₅₀ = 5 μM), CoCl₅₀ = 58 μM) and retained at a fixed concentration of external sodium indicating the involvement of Ca²⁺ channels rather than Na⁺/Ca²⁺ exchange in swelling-induced Ca²⁺ influx. The populations of calcium channels observed in hypoosmotic and depolarizing conditions are different in three aspects: (i) kinetics of ⁴⁵Ca²⁺ entry; (ii) insensitivity to dihydropyridines and ω-conotoxin GVIA; (iii) insensitivity to preliminary depolarization by high potassium. The effects of swelling and depolarization on Ca²⁺ uptake were additive. No change in membrane potential monitored with diS-C₃-(5) was recorded during synaptosome hypotonic swelling. The results suggest the existence in synaptosomal plasma membrane of volume-dependent calcium-permeable channels with properties distinct from those of the voltage-dependent calcium channels. Activation of these channels may constitute an early event in volume regulation of nerve terminals in anisoosmotic conditions.     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Characterization of pancreatic somatostatin binding sites with a I-somatostatin 28 analog

Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S₂₈): ¹²⁵I-[Leu⁸, DTrp²²,Tyr²⁵] S₂₈. The binding was highly dependent on calcium ions. In 0.2 mM free Ca²⁺ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (K_D=0.05±0.01 nM, B_max=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca²⁺ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA^1,2[AzaAla^4–5,DTrp⁸,Phe^12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using ¹²⁵I-[Leu⁸,DTrp²²,Tyr²⁵]S₂₈ as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand ¹²⁵I-[Tyr¹¹]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors.     

Insulin secretion and intracellular Ca rises in monolayer cultures of neonatal rat β-cells

Glucose-induced insuline release, glucose-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i), and voltage-dependent Ca²⁺ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca²⁺]_i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca²⁺ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca²⁺]₂ rise and, like deprivation of extracellular Ca²⁺, prevented the glucose-induced rise in [Ca²⁺]_i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca²⁺ channels was demonstrated directly by measuring Ca²⁺ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca²⁺]₁ after membrane depolarization by 45 mMm K⁺ or 200 μM tolbutamide. Thus, in cultured β-cells of 3–5 day-old rats the coupling of glucose stimulation to Ca²⁺ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.     

Slippage of η-allenyl/propargyl to an η structure and tautomerization of η-propargyl to η-allenyl in ligand addition and substitution reactions of platinum(II) complexes

Reactions of [(PPh₃)₂Pt(η³-CH₂CCPh)]OTf with each of PMe₃, CO and Br⁻ result in the addition of these species to the metal and a change in hapticity of the η³-CH₂CCPh to η¹-CH₂CCPh or η¹-C(Ph)=C=CH₂. Thus, PMe₃ affords [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]⁺, CO gives both [trans-(PPh₃)₂Pt(CO)(η¹-CH₂CCPh)]⁺ and [trans-(PPh₃)₂Pt(CO)(η¹-C(Ph)=C=CH₂)]⁺, and LiBr yields cis-(PPh₃)₂PtBr(η¹-CH₂CCPh), which undergoes isomerization to trans-(PPh₃)₂PtBr(η¹-CH₂CCPh). Substitution reactions of cis- and trans-(PPh₃)₂PtBr(η¹-CH₂CCPh) each lead to tautomerization of η¹-CH₂CCPh to η¹-C(Ph)=C=CH₂, with trans-(PPh₃)₂PtBr(η¹-CH₂CCPh) affording [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]⁺ at ambient temperature and the slower reacting cis isomer giving [trans-(PPh₃)(PMe₃)₂Pt(η¹-C(Ph)=C=CH₂)]⁺ at 54 °C . All new complexes were characterized by a combination of elemental analysis, FAB mas spectrometry and IR and NMR (¹H, ¹³C{¹H} and ³¹P{¹H}) spectroscopy. The structure of [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]BPh₄·0.5MeOH was determined by single-crystal X-ray diffraction analysis.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.     

Characterization of the novel CCK analogs JMV-180, JMV-320, and JMV-332 in H345 cells

The interaction of the novel CCK analogs JMV-180, JMV-320, and JMV-332 with CCK-B/gastrin receptors on small cell lung cancer (SCLC) cells was investigated. JMV-180, JMV-320, and JMV-332 potently inhibited specific binding of ¹²⁵I-CCK-8 to CCK-B/gastrin receptors expressed on the SCLC cell line NCI-H345 (H345) with IC₅₀ values of 4.9, 1.8, and 7.0 nM, respectively. JMV-320 and JMV-332 stimulated intracellular calcium ([Ca²⁺]_i) release in a dose-dependent manner in cells preloaded with indo-1. JMV-180 did not stimulate [Ca²⁺]_i but inhibited the [Ca²⁺]_i release elicited by 10 nM CCK-8 in a dose-dependent manner. These data indicate that JMV-320 and JMV-332 function as CCK-B/gastrin receptor agonists while JMV-180 functions as a CCK-B/gastrin receptor antagonist in H345 cells.     

Binding of divalent copper and calcium ions to poly I

The effect ot Cu²⁺ and Ca²⁺ ions, on the ultraviolet differential (UVD) spectra of single-stranded poly I was studied and the coordination (Δε_b) and conformation (Δε_c) conponents of the spectra calculated The comparison of Δε_b and the UVD spectrum of protonated IMP leads to the conclusion that N(7) ot inosine-5'-monophosphate (IMP) is a coordinating site tor Ca²⁺ and Cu²⁺ ions on the polymer bases. The binding ot Ca²⁺ and Cu²⁺ ions causes differently directed displacements of the four absorption bands of poly I, which are observed in the wavenumber range (50-34) × 10³ cm⁻¹ The calculation of concentration dependencies tor the association constants (K“) ot Ca²⁺ and Cu²⁺ ions binding to poly I bases shows that the binding is cooperative The K“ values for the poly I + Ca²⁺ complex are two orders of magnitude lower than those for the poly 1 + Cu²⁺ complex At low ion concentrations, binding to the poly I phosphates predominates and increases the degree of the polynucleotide helicity. At higher concentrations the spectra are mainly affected by the ion binding to bases, which results in melting of the helical parts of poly I At Ca²⁺ concentrations exceeding 10⁻³ M light-scattering aggregates are formed. The degree of monomer order in them is close to that observed in multistranded helices of poly I     

Two bombesin analogues discriminate between neuromedin B- and bombesin-induced calcium flux in a lung cancer cell line

We examined the profile of two bombesin (BN) antagonists, (CH₃)₂CHCO-His-Trp-Ala-Val- -Ala-His-Leu-NHCH₃] (ICI 216140) and [ -Phe⁶,des-Met¹⁴]BN(6–14)ethylamide (DPDM-BN EA), against neuromedin B-induced Ca²⁺ mobilization in the small cell lung cancer (SCLC) line NCI-H345. Neuromedin B (NMB), a BN-like peptide sharing sequence homology with ranatensin, elicited a concentration-dependent Ca²⁺ release (in part) from intracellular stores. Sequential addition of NMB attenuated Ca²⁺ mobilization. Desensitization occurred between BN and NMB; depletion of intracellular Ca²⁺ is a likely mechanism because thapsigargin stimulated Ca²⁺ release after a maximally desensitizing dose of NMB. ICI 216140 and DPDM-BN EA competitively inhibited BN-induced Ca²⁺ transients. In contrast, these compounds antagonized NMB-stimulated Ca²⁺ transients in a noncompetitive manner. The pharmacological profiles obtained support receptor heterogeneity for BN-like peptides on this SCLC line, underscoring the need for thorough examination of dose-response relationships when investigating effects of BN analogues on intact cells.     

2, 5-Di(Tert-Butyl)-1, 4-Benzohydroquinone —A Novel Mobilizer Of The Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Pool

Isolated hepatocytes and the isolated perfused rat liver have been used to study the alterations of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) produced by 2,5-di(tert-butyl)-l.4-benzohydroquinone (tBuBHQ), a potent inhibitor of hepatic microsomal Ca²⁺ sequestration (Moore. G.A., McConkey. D.J., Kass, G.E.N., OBrien, P.J. and Orrenius, S. FEBS Lett.,224, 331-336). (1987). Addition of tBuBHQ to isolated hepatocytes caused a rapid increase in [Ca²⁺]_i which was similar in magnitude to the [Ca²⁺]_i elevation induced by the Ca²⁺ mobilizing hormone, vasopressin. In contrast with vasopressin which caused a Ca²⁺ transient, tBuBHQ elevated [Ca²⁺]_i to a new steady state that was maintained for up to 15-20min. When vasopressin was administered during the tBuBHQ-induced period of elevated [Ca²⁺]_i. [Ca²⁺]_i, rapidly returned to basal levels. Similarly, if vasopressin was administered just prior to tBuBHQ, the resultant tBuBHQ-dependent change in [Ca²⁺]_i was transient. and not sustained. The hydroquinone mobilized the same intracellular Ca²⁺ pool as inositol 1,4,5-trisphosphate. but tBuBHQ did not produce any detectable inositol polyphosphate accumulation. IBuBHQ stimulated glucose release from perifused hepatocytes. mimicking the effect of vasopressin. In the perfused liver, tBuBHQ infusion produced a single, slow and prolonged release of Ca²⁺ into the perfusate and inhibition of subsequent vasopressin-induced Ca²⁺ effluxes. Inhibition of the response to vasopressin was reversed over time, and closely correlated with the extent of inhibition of both Ca²⁺ sequestration and (Ca²⁺-Mg²⁺)-ATPase activity in microsomes isolated from the isolated perfused liver. The present results are consistent with tBuBHQ inhibiting ATP-dependent Ca²⁺ sequestration by a direct effect on the endoplasmic reticular Ca²⁺ pump, which results in net Ca²⁺ release and elevation of [Ca²⁺]_i. Furthermore. vasopressin appears to stimulate active removal of increased [Ca²⁺] from the hepatocyte cytosol by a mechanism which does not depend on reuptake of Ca²⁺ into the endoplasmic reticulum

2,5-Di(tert-butyl) -l,4-benmhydroquinone. calcium. hepatocytes. perfused liver, endoplasmic reticulum     

Phosphorylation of an actin · tropomyosin · troponin complex from human skeletal muscle

A human skeletal actin · tropomyosin · troponin complex was phosphorylated in the presence of [γ-³²P]ATP, Mg²⁺, adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 μM cyclic AMP. In the presence of 10⁻⁷ M Ca²⁺ and protein kinase 0.1 mole of [³²P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [³²P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca²⁺, 5 · 10⁻⁵ M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [³²P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [³²P]phosphate. Increasing the Ca²⁺ concentration did not significantly decrease the [³²P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstituted human skeletal actomyosin made with the [³²P]phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca²⁺.     

Release of azurophilic granule contents in fMLP-stimulated neutrophils requires two activation signals, one of which is a rise in cytosolic free Ca

We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca²⁺ ([Ca²⁺]_i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the β-glucuronidase substrate 4-methylumbelliferyl-β- -glucuronide. We found that the transient rise in [Ca²⁺]_i induced by fMLP is a necessary signal to obtain to obtain maximal degranulation. When this Ca²⁺ transient is prevented by the Ca²⁺ chelator BAPTA, degranulation can still be induced by a stimulated Ca²⁺ influx, albeit to a lower extent. We also studied the degranulation process in the neutrophils of a patient with a generalized chemotactic defect. Release of β-glucuronidase from the patient's neutrophils could not be induced despite the occurrence of a normal Ca²⁺ response and normal degranulation of specific granules. We conclude that, besides an increase in [Ca²⁺]_i], an additional signal is required for the fusion of azurophilic granules with the plasma membrane in human neutrophils.     

Dissociation of Intracellular Ca Release and Ca Entry Response to 5-Hydroxytryptamine in Cultured Canine Tracheal Smooth Muscle Cells

The relationship between the agonist-sensitive Ca²⁺ pool and those discharged by the Ca²⁺-ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca²⁺ ([Ca²⁺]_i), followed by a sustained plateau phase that was dependent on extracellular Ca²⁺. In such cells, TG produced a concentration-dependent increase in [Ca²⁺]_i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca²⁺. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca²⁺]_i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca²⁺-ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca²⁺-influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca²⁺ stores is sufficient for activation of Ca²⁺ influx. Some characteristics of the Ca²⁺-influx activated by depletion of internal Ca²⁺ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca²⁺ influx was inhibited by La³⁺, membrane depolarisation, and the novel Ca²⁺-influx blocker 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca²⁺ influx by TG also was blocked by La³⁺, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca²⁺ stores in TSMCs is sufficient for activation of Ca²⁺ influx, and (2) the agonist-activated Ca²⁺ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool are indistinguishable.     

Dihydropyridine-sensitive Ca channel in aneurally cultured human muscles Relationship between high-affinity binding site and inhibition of calcium uptake

Dihydropyridine-sensitive Ca²⁺ channels and the relationship between binding of dihydropyridine derivatives and depolarization-induced Ca²⁺ uptake have been studied in aneurally cultured human muscle. Analysis of the equilibrium binding of the 1,4-dihydropyridine derivative (+)-PN200-110 revealed a single high-affinity binding site with a K_d of 0.15±0.05 nM and a B_max of 87±12 fmol/mg protein. Inhibition of (+)-[³H]PN200-110 binding by nitrendipine revealed a K_i of 0.8 nM for the nitrendipine-receptor complex. Depolarization of cultured human muscle achieved by elevating the K⁺ concentration increased the uptake ⁴⁵Ca²⁺ which was inhibited by nitrendipine with an IC₅₀ of 1.1 nM. This study demonstrates that aneurally cultured human muscle has dihydropyridine-sensitive voltage-dependent Ca²⁺ channels which are functional when the fibers are depolarized.     

Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca²⁺ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC₅₀) and maximal inhibition of BK induced an increase in [Ca²⁺]_i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca²⁺ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K_D and B_max of the BK receptor for binding (control: K_D = 1.7 ± 0.2 nM; B_max = 47.3 ± 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca²⁺ mobilisation in VSMCs.