Requirement of Ca2+ for aflatoxin production: inhibitory effect of Ca2+ channel blockers on aflatoxin production by Aspergillus parasiticus NRRL 2999

Aflatoxin production by Aspergillus parasiticus NRRL 2999 was inhibited when Ca2+ channel blockers, i.e., verapamil and diltiazem (> 1 mmol 1(-1)), were included in the culture medium. Inhibition was not accompanied by growth inhibition, nor was the [14C]-glucose uptake by the organism altered. However, both the compounds inhibited [14C]-acetate incorporation into aflatoxin B1 in a dose-dependent manner and decreased sporulation of the organism. Even though a nutritional role for Ca2+ has not been demonstrated unequivocally in fungi, the present study suggests the importance of Ca2+ in the production of these secondary metabolites.     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.     

Synthesis of phosphatidylcholines in rat hepatocytes. Possible regulation by norepinephrine via an alpha-adrenergic mechanism

The effect of norepinephrine on phosphatidylcholine and phosphatidylethanolamine formation was investigated in short-term incubations with freshly isolated rat hepatocytes. In the presence of dl-propranolol, norepinephrine decreases the incorporation of [methyl-14C]choline into phosphatidylcholines in a dose-dependent manner. At a concentration of 50 microM, norepinephrine (plus 20 microM propranolol) inhibits the incorporation of [methyl-14C]choline over a wide range of choline concentrations (59% inhibition at 5 microM choline; 34% inhibition at 1 mM choline). Norepinephrine also decreases the incorporation rates of [1-14C]palmitic acid and [1-14C]oleic acid into phosphatidylcholines. The effect of norepinephrine is mediated through an alpha-adrenergic receptor. Norepinephrine (plus propranolol) does not decrease the uptake or phosphorylation rate of [methyl-14C]choline. Pulse-label and pulse-chase studies indicate that the conversion rate of phosphocholine to CDP-choline, catalyzed by CTP:phosphocholine cytidylyltransferase, is diminished by norepinephrine. In contrast with the inhibitory effect of norepinephrine on phosphatidylcholine synthesis, this hormone stimulates the formation of phosphatidylethanolamines from [1,2-14C]ethanolamine. This increased incorporation rate is apparent at ethanolamine concentrations above 25 microM. A combination of norepinephrine and propranolol decreases, however, the synthesis of phosphatidylcholines from [1,2-14C]ethanolamine. The results indicate that alpha-adrenergic regulation dissociates the synthesis of phosphatidylcholines from that of phosphatidylethanolamines.     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca2+ and calmodulin

The effects of Ca2+ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [32P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca2+. Maximal incorporation of [32P] into the protein was achieved with free Ca2+ concentrations of 90 microM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 microM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [32P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca2+ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca2+ concentrations between 10(-6) - 10(-5) M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca2+ and calmodulin.     

Inhibition by chloroquine of the secretion of very low density lipoproteins by cultured rat hepatocytes

Cultured rat hepatocytes were incubated in medium containing 1.0 mM oleic acid. The incorporation of [3H]glycerol into cell-associated and medium triacylglycerols was measured after 2 h incubation. More than 95% of the secreted [3H]triacylglycerols were recovered in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Chloroquine and other lysosomotropic amines promoted a marked decrease in [3H]triacylglycerol secretion from the hepatocytes while the synthesis was unaffected. At 50-200 microM final concentration, chloroquine inhibited secretion of triacylglycerols by 70-90% of the control. Similar results were obtained when the mass of secreted triacylglycerols was measured. Chloroquine caused decreased secretion of [3H]triacylglycerols after 15-30 min incubation and the inhibitory effect was completely reversible within 1-2 h after washout of chloroquine. The reduced triacylglycerol secretion was not due to increased reuptake of secreted lipoproteins or decreased protein synthesis caused by chloroquine. Electron microscopy of chloroquine-treated cells showed that the inhibition of VLDL secretion occurs at or prior to the level of the Golgi apparatus. These results suggest that chloroquine interferes with crucial steps in the secretory process and/or that lysosomal function could be essential for secretion of VLDL.     

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.     

Inhibition of phosphatidylcholine synthesis by vasopressin and angiotensin in rat hepatocytes.

The addition of 1 microM-vasopressin or -angiotensin to isolated rat hepatocytes induced a fast transient inhibition of the rate of incorporation of [Me-3H]choline into phosphatidylcholine. The cationophore A23187 induced a similar inhibition of phosphatidylcholine synthesis. The addition of micromolar Ca2+ to rat liver microsomes inhibited the activity of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase. This inhibition is due a decrease in the Vmax. of the enzyme without affecting the Km for CDP-choline. It is concluded that Ca2+ regulates phosphatidylcholine synthesis in rat liver.     

Increased lung uptake of liposomes coated with polysaccharides

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na+), or in a medium containing only 25 mM Na+. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin ( PzO ) is not affected. Amiloride also inhibits the carbachol-induced 45Ca efflux from rabbit pancreatic acini, but again not that induced by PzO . The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and 45Ca efflux are 40 and 80 microM, respectively. Amiloride also competitively inhibits the specific binding of [3H]quinuclidinyl benzylate ( [3H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Apolipoprotein synthesis and secretion in Hep G2 cells: effects of monensin and cycloheximide.

Hep G2 cells were used to study the relationship between apolipoprotein synthesis and secretion, as revealed by their interaction with agents modulating these processes. Cycloheximide inhibited the secretion of both apolipoproteins (apo) AI and B, but the reduction in apo AI secretion was evident at earlier times. Monensin also inhibited secretion of apo AI and apo B, but only apo AI accumulated intracellularly. Pulse-chase studies showed that, at concentrations of monensin that had no effect on total protein synthesis, apo B synthesis was specifically inhibited. Triacylglycerol synthesis was inhibited to the same extent as apo B synthesis, but this preceded the latter inhibition and unlike apo B there was an accumulation of intracellular triglyceride. These results suggest that distinctive mechanisms modulate the synthesis and secretion of apo AI and apo B, and that apo B synthesis can be specifically inhibited by mechanisms that initially block triglyceride production.     

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.     

Guanine nucleotide effects on catecholamine secretion from digitonin-permeabilized adrenal chromaffin cells

The nonhydrolyzable GTP analogue guanosine 5'-(beta, gamma-imido)triphosphate (GMP-PNP) produced an ATP-dependent but Ca2+-independent stimulation of [3H]norepinephrine release from permeabilized chromaffin cells. This stimulation of secretion was 25-35% of the secretion induced by 10 microM Ca2+. A similar Ca2+-independent stimulation was produced by other non-hydrolyzable GTP analogues. No effect was seen with a variety of other nucleotides, including GTP. The GMP-PNP effect was specifically inhibited by low concentrations of guanine nucleotides. Addition of cAMP did not mimic the Ca2+-independent GMP-PNP effect, but did slightly enhance Ca2+-dependent secretion. Pretreatment with pertussis toxin had no effect on Ca2+-dependent secretion or on the GMP-PNP effect. There was no detectable diglyceride or inositol phosphate produced during GMP-PNP treatment, and addition of diglyceride and inositol trisphosphate did not induce secretion. Guanosine 5'-(beta-thio)diphosphate (GDP-beta-S), in addition to its ability to inhibit the GMP-PNP effect, partially inhibited Ca2+-dependent secretion. At 10 microM free Ca2+, the effects of GMP-PNP and Ca2+ were nonadditive. In fact, secretion in the presence of both GMP-PNP and 10 microM Ca2+ was slightly less than secretion due to Ca2+ alone. These data suggest that a guanine nucleotide-dependent process interacts in some way with one or more components of the normal Ca2+-dependent secretory pathway. However, it may not be an intrinsic part of the mechanism underlying Ca2+-dependent secretion.     

Tricyclic antidepressants inhibit voltage-dependent calcium channels and Na(+)-Ca2+ exchange in rat brain cortex synaptosomes

We have used a resting (5 mM K+) or depolarizing (60 mM K+) choline-based medium, and a nondepolarizing sodium-based or choline-based medium, to characterize the inhibitory potential of tricyclic antidepressants against the voltage-dependent calcium channels or the Na(+)-Ca2+ exchange process, respectively, in synaptosomes from rat brain cortex. Imipramine, desipramine, amitriptyline, and clomipramine inhibited net K(+)-induced 45Ca uptake with similar IC50 values (26-31 microM), and this uptake was also inhibited by diltiazem with an IC50 of 36 microM; these results indicate an inhibition of voltage-dependent calcium channels by tricyclic antidepressants. The net uptake of 45Ca induced by Na(+)-Ca2+ exchange was also inhibited by the four tricyclic antidepressants tested, but not by diltiazem; imipramine (IC50 = 94 microM) was a more potent inhibitor of this process than desipramine (IC50 = 151 microM), and the IC50 values of amitriptyline (107 microM) and clomipramine (97 microM) were similar to that of imipramine. Some degree (approximately 25%) of brain calcium channel blockade could be present at the steady-state concentrations of tricyclic antidepressants expected to occur therapeutic use of these compounds to treat depression or panic disorder.     

Insulin effects on apolipoprotein B production by normal, diabetic and treated-diabetic rat liver and cultured rat hepatocytes.

1. The effect of insulin on apolipoprotein (apo B) secretion was studied in 24 h recirculating liver perfusions of isolated normal, diabetic and insulin-treated diabetic rats. In single perfusions from each group apo B accumulated in the media in a linear fashion. 2 In perfusions of normal rat livers, when the medium contained insulin plus cortisol, apo B production was significantly inhibited (by 35.8%), demonstrating a hormone effect on apo B secretion. 3. In perfusions of diabetic-rat livers, apo B production was decreased to 11.8% of normal when the medium contained no hormones, and was not significantly changed by the addition of insulin plus cortisol to the medium, suggesting that the hormone effect on apo B secretion is missing in long-term hypoinsulinaemic states. 4. Treatment of diabetic rats with daily insulin injection restored apo B production and restored the effect of insulin plus cortisol in the medium to inhibit apo B secretion during perfusion. 5. Parallel studies of apo B secretion with insulin alone, cortisol alone and insulin plus cortisol in the medium were performed in primary cultures of hepatocytes to compare results from liver perfusions. 6. Apo B secretion by hepatocytes from normal, diabetic and treated-diabetic rats was inhibited (by 36.8%, 57.1% and 57.9% respectively) when insulin alone was added to the medium. 7. Insulin plus cortisol inhibited apo B secretion by hepatocytes from normal and treated diabetic rats (by 30.2% and 47.2% respectively), but failed to inhibit apo B secretion by hepatocytes from diabetic rats.     

Effect of (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55,940) on intracellular Ca2+ levels in human osteosarcoma cells

The study was undertaken to explore the effect of CP55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a drug commonly used as a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in MG63 human osteoblast-like epithelial cells. [Ca2+]i was measured in suspended cells by using the fluorescent dye fura-2 as an indicator. At concentrations between 2-20 microM, CP55,940 increased [Ca2+]i in a concentration-dependent manner with an EC50 of 8 microM. The [Ca2+] signal comprised an initial rise, a slow decay, and a sustained phase. CP55940 (10 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists (AM-251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole3-carboxamide; AM-281, 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide). Extracellular Ca2+ removal decreased the maximum value of the Ca2+ signals by 50%. CP55,940 induced quench of fura-2 fluorescence by Mn2+ (50 microM), suggesting the presence of Ca2+ influx across the plasma membrane. CP55,940 (10 microM)-induced [Ca2+]i increase in Ca(2+)-free medium was inhibited by 84% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 10 microM CP55,940 in Ca(2+)-free medium abolished thapsigargin-induced [Ca2+]i increase. At 1 microM, nifedipine, verapamil, and diltiazem did not alter CP55, 940 (10 microM)-induced [Ca2+]i increase. CP55,940 (20 microM)-induced Ca2+ release was not affected when phospholipase C was inhibited by 2 microM U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione). CP55,940 (20 microM) did not induce acute cell death after incubation for 30 min as assayed by trypan blue exclusion. Collectively, CP55,940 induced significant [Ca2+]i increases in osteoblasts by releasing store Ca2+ from thapsigargin-sensitive stores and by causing Ca2+ entry. The CP55,940's action appears to be independent of stimulation of CB1 cannabinoid receptors.     

Bile acids inhibit secretion of very low density lipoprotein by rat hepatocytes

The influence of taurocholate on very low density lipoprotein (VLDL) triacylglycerol synthesis and secretion was studied by isolated rat liver-parenchymal cells. The incorporation of [3H]glycerol into cell-associated and VLDL triacylglycerols were measured after incubation in medium containing 0.75 mM oleate. Taurocholate caused a maked decrease in VLDL [3H]triacylglycerol secretion from the hepatocytes: 50-150 microM taurocholate inhibited secretion of VLDL [3H]triacylglycerols by 70-90%. Similar results were obtained when the mass of secreted VLDL triacylglycerols was measured. Taurocholate caused a decreased secretion of VLDL [3H]triacylglycerols after 15-30 min incubation. A higher amount of cellular triacylglycerols was found in taurocholate-supplemented cells. Furthermore taurocholate did not change the intracellular lipolysis of triacylglycerols. These results suggest that bile acids interfere more probably with the assembly and/or secretion of VLDL-particles and not with earlier stages of VLDL formation, e.g. triacylglycerol synthesis.     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Characterization of the Ca2+ coordination site regulating binding of Ca2+ channel inhibitors d-cis-diltiazem, (+/-)bepridil and (-)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes

Ca2+ inhibits (-)[3H]desmethoxyverapamil, d-cis-[3H]diltiazem and (+/-)[3H]bepridil binding to skeletal muscle transverse-tubule membranes with a half-maximum inhibition constant, K0.5 = 5 +/- 1 microM. This value is close to that of the high affinity Ca2+ binding site which controls the ionic selectivity of the Ca2+ channel found in electrophysiological experiments suggesting that the Ca2+ coordination site which regulates the ionic selectivity is also the one which alters binding of the Ca2+ channel inhibitors investigated here. Ca2+ and (-)D888 bind to distinct sites. Occupation of the Ca2+ coordination site decreases the affinity of (-)D888 for its receptor by a factor of 5. Other divalent cations have the same type of inhibition behavior with the rank order of potency Ca2+ (K0.5 = 5 microM) greater than Sr2+ (K0.5 = 25 microM) greater than Ba2+ (K0.5 = 50 microM) greater than Mg2+ (K0.5 = 170 microM).     

Clinical potency of calcium channel blockers in asthma may be related to their inhibition of receptor-mediated phasic responses in vitro.

The calcium channel blockers (CCB) have been clinically effective in exercise-induced asthma. The completeness of protection with the CCB might be related specifically to inhibition of Ca2+ influx or release. To examine this hypothesis, the rank order of potency of inhibition of the CCB, nicardipine, diltiazem and verapamil on the steady-state and kinetic parameters of the phasic and tonic responses to the muscarinic receptor agonist carbachol (10 microM) and KCl (40 mM) in the intact isolated guinea-pig trachea was determined. The Ca2+ channel agonist Bay K 8644 was also examined for its effects on intracellular Ca2+. Nicardipine abolished the KCl response at both 0.1 microM and 1 microM concentrations. The amplitude of the KCl response was inhibited equally by 1 microM diltiazem (61% inhibition) and 1 microM verapamil (68% inhibition). The rate constant of onset of the KCl response was similarly inhibited 60% by diltiazem and 66% by verapamil. Nicardipine abolished the carbachol phasic response at the 1 microM concentration. The amplitude of the phasic response was inhibited equally by 0.1 microM nicardipine (61.3% inhibition), 1 microM diltiazem (64.5% inhibition) and 1 microM verapamil (71% inhibition). The rate constant of decay of the phasic response was inhibited equally by 0.1 microM nicardipine (43% inhibition) and 1 microM diltiazem (29% inhibition). The rate constant of onset of the phasic response was unaffected by nicardipine, diltiazem and verapamil. Only 1 microM nicardipine inhibited the amplitude and rate constant of onset of the tonic response. The only effect of Bay K 8644 (1 microM) was to increase the phasic response amplitude. The CCB demonstrate a similar order of potency for inhibition of the phasic responses and clinical efficacy of the CCB in exercise-induced asthma (nicardipine > verapamil > diltiazem).(ABSTRACT TRUNCATED AT 250 WORDS)