Evidence for a dipeptide transport system in renal brush border membranes from rabbit

Papain treatment of renal brush border vesicles was carried out as a successful first step towards the purification of the membrane components involved in dipeptide transport. The treated vesicles exhibited increased specific transport activity of glycyl-l-proline. In contrast, the specific transport activity of l-alanine in the treated vesicles was less than that in the control vesicles. Papain treatment resulted in the solubilization of 38% of protein, 55% of alkaline phosphatase, 90% of γ-glutamyltransferase and 95% of leucine aminopeptidase. There was no change in the intravesicular volume nor was there any increase in vesicular permeability. Glycyl-l-proline transport was Na⁺-independent in the control and papain-treated vesicles. Diamide reduced the Na⁺-dependent l-alanine transport while glycyl-l-proline transport remained unaffected in the presence of Na⁺. Many dipeptides inhibited glycyl-l-proline transport both in the presence and absence of Na⁺. The inhibition by dipeptides was greater than the inhibition by equivalent concentrations of free amino acids. These data demonstrate that renal brush border vesicles can efficiently handle dipeptides by a mechanism completely different from that of amino acid transport.     

Effect of pH on the transport of Krebs cycle intermediates in renal brush border membranes

Lowering extravesicular pH stimulated Na⁺-dependent citrate transport in renal brush border membrane vesicles: e.g., at pH_out = 5.5, the initial rate of citrate uptake was increased 10-fold compared to parallel control experiments at pH 7.5. The same experimental conditions had little effect on succinate uptake. The influence of pH on citrate transport is a product of the extravesicular H⁺ concentration; pH gradients did not potentiate the effects nor were proton gradients capable of driving transport in the absence of Na⁺. The effect of pH is adequately explained if only the mono- and divalent species of citrate (Cit^1?, Cit^2?) are considered acceptable substrates for transport. The stimulatory influence of pH on transport correlated quite well with pH-related increases in the concentrations of Cit^1? and Cit^2?, and over the same pH range [Cit^3?] was inversely related to citrate uptake. A model of the Na⁺-dependent dicarboxylate transport system is discussed in which three sodium ions are translocated per molecule of dicarboxylic acid.     

Effect of phospholipid methylation on calcium transport and (Ca2+ + Mg2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na⁺ + K⁺)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na⁺ + K⁺)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with $\text{S-}\text{adenosyl}\text{[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, $\text{S-}\text{adenosylmethionine}$, exhibited increased (12–15%) (Ca²⁺ + Mg²⁺)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ and not due to a change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Ca²⁺. Preincubation of membranes with $\text{S-}\text{adenosylhomocysteine}$, a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.     

Diuretic effects on renal brush border membrane transport and metabolism

Previous studies have indicated that the thiazide diuretics exert effects on proximal electrolyte transport. To determine whether the locus of these effects is at the brush border membrane (BBM) and if renal metabolism is affected, adult female Sprague-Dawley rats were acutely treated with either 1 mg/kg metolazone, 20 mg/kg chlorothiazide followed by a 20 mg/kg/hr maintenance infusion, 10 mg/kg acetazolamide followed by a 10 mg/kg/hr maintenance infusion, or the vehicles only. Administration of these agents resulted in an approximately tenfold increase in sodium excretion. Neither urinary phosphate nor inulin excretion changed significantly in any group. Sodium dependent BBM vesicle phosphate transport was examined at 0.15, 0.5, and 1 and 120 minute incubation periods in the diuretic treated groups and their respective control groups. Decreased uptake was seen in all pre-equilibrium time points in rats treated with metolazone: 0.15 minutes: 221 +/- 24 pmoles/mg protein (pmol/mg prot) in control rats versus (vs) 185 +/- 23 pmoles/mg prot in metolazone-treated animals (P less than .05) ; 0.5 minutes: 463 +/- 54 vs 369 +/- 49 pmol/mg prot (P less than .005); 1 minute: 549 +/- 74 vs 460 +/- 61 pmol/mg prot (P less than .05); no significant difference in phosphate transport was noted at the two hour equilibrium time point. No significant differences in sodium dependent phosphate transport existed between chlorothiazide or acetazolamide treated rats and control animals. Substrate-stimulated renal gluconeogenesis did not differ between metolazone treated and control animals. We therefore conclude that metolazone inhibits phosphate transport through an effect on the BBM and does not affect renal gluconeogenesis in the rat.     

Effect of anions on folate binding by isolated brush border membranes from rat kidney

The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3′, 5′, 9-³H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70–75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3′,5′,9-³H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroylglutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroylglutamic acid. Inhibition of [3′,5′,9-³H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3′,5′,9-³H]pteroylglutamic acid.     

Na+-dependent,potential-sensitive l-ascorbate transport across brush border membrane vesicles from kidney cortex

l-Ascorbate is taken up into brush border vesicles from kidney cortex of rat, rabbit and guinea pig by an efficient, Na⁺-dependent and potential-sensitive transport process. This uptake shows saturation ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>:0.1–0.3\; mM</mtext>}}$) and is strongly stimulated by low concentrations of N₃^?. Erythorbate (d-isoascorbate) seems to be another, but poorer, substrate of the same transporter.     

K+- and Na+-gradient-dependent transport of l-phenylalanine by mouse intestinal brush border membrane vesicles

In the presence of an Na⁺- or a K⁺-gradient ($\text{outside > inside}$), l-phenylalanine uptake exhibited an overshoot phenomenon indicating active transport. The amplitudes of the overshoots were increased by increasing either Na⁺ or K⁺ concentrations in the incubation media, indicating that binding alone cannot account for the K⁺ effect. The K⁺-induced overshoot is not due to the presence of a membrane potential alone, as a gradient of choline chloride failed to produce it. Li⁺ could also substitute for Na⁺ though less potent than Na⁺ in inducing an overshoot. Uptake of l-leucine also showed Na⁺- and K⁺-effects and l-leucine and l-alanine could inhibit the Na⁺- and K⁺-overshoots obtained with phenylalanine. These results lead us to postulate the presence of a carrier for neutral amino acids dependent on monovalent cation with higher affinity for Na⁺ in mouse intestine. The Na⁺- and K⁺-driven active transport of l-phenylalanine were shown to be dependent on the presence of a membrane potential, as short-circuiting the membrane with FCCP reduced the amplitude of the overshoots seen with both ions. However, substitution of Cl^? by more lipophilic anions (NO₃^?, SCN^?) produced an inhibition of uptake. A preliminary analysis of the interrelations between Na⁺ and K⁺ for l-phenylalanine uptake showed complex interactions which can be best explained by mutual competition for a common carrier at both sides of the membrane. These results suggest the presence of a new transport system or a variant of an ASC-type system for l-phenylalanine (and neutral amino acids) in the mouse intestine. However, our studies do not rule out the possible involvement of more than one system for neutral amino acid uptake.     

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes.

Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the ³²P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of ³²P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent K_m for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10^?8m cAMP. The K_a values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.     

The activity of kidney lysosomal hydrolases and renin in hypertensive rats.

In renal cortices of hypertensive rats the activity of renin and β-glucuronidase is significantly enhanced, while the activity of acid phosphatase remains pratically unchanged. A significant decrease in protein content accompanies the rise in enzyme activity. The distribution pattern of renin, β-glucuronidase and acid phosphatase after isopycnic centrifugation of cortical homogenates from hypertensive animals is different from that of controls.     

Hormonal regulation of ornithine aminotransferase biosynthesis in rat liver and kidney.

The relative rates of ornithine aminotransferase (OAT) synthesis in vivo were studied by pulse-labeling rats with [4,5-³H]leucine, isolating the mitochondrial enzyme protein by immunoprecipitation with a monospecific antibody, dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels, and determining the radioactivity in OAT. After 4 days of treatment with triiodothyronine (T₃), both the enzyme activity level and the relative synthetic rate of OAT in rat kidney were elevated over twofold. The level of hepatic OAT activity was unaffected by this treatment. Thyroidectomy caused a 50% drop in the basal level of OAT activity and synthesis in kidney but not in liver. Although the basal levels of activity and synthesis of both renal and hepatic OAT were unaffected by adrenalectomy, the glucagon induction of the enzyme in liver was enhanced by about one-third and the T₃ induction in kidney was suppressed 50% by this operation. After 4 days of treatment with estrogen, both the enzyme activity level and the relative synthetic rate of OAT in male rat kidney were elevated nearly 10-fold. Hepatic OAT activity and synthesis were unaffected by this regimen. Thyroidectomy almost completely abolished the estrogen induction of OAT in kidney. OAT induction by estrogen could be restored by treating thyroidectomized rats with T₃. Simultaneous administration of T₃ plus estrogen to intact rats produced a multiple effect, resulting in a striking 20-fold induction of renal OAT. Although administration of either T₃ or estrogen causes an increase in the synthesis of immunoprecipitable OAT protein in rat kidney, each of these hormones may induce OAT by a different mechanism.     

Effect of parathyroid hormone on Na+-dependent phosphate transport and cAMP-dependent 32P phosphorylation in brush border vesicles from isolated perfused canine kidneys

Concentrative uptake of 32Pi induced by the dissipation of a Na+ gradient (overshoot) was demonstrated in brush border membrane vesicles obtained from isolated perfused canine kidneys. Na+-dependent 32Pi transport was decreased in brush border vesicles from isolated kidneys perfused with parathyroid hormone (PTH) for 2 h compared to uptake measured in vesicles from kidneys perfused without PTH. Cyclic AMP-dependent 32P phosphorylation of a 62,000 Mr protein band was demonstrable on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of membrane suspensions from kidneys perfused +/- PTH. Evidence that perfusion with PTH resulted in cAMP-dependent phosphorylation in isolated kidneys from parathyroidectomized dogs (decreased cAMP-dependent 32P phosphorylation of the 62,000-Mr band in brush border vesicles) was obtained after 2-h perfusion with PTH. Decreased 32P phosphorylation was not observed if membranes were allowed to dephosphorylate prior to 32P phosphorylation in vitro. We conclude that brush border vesicles from isolated perfused canine kidneys can be used to study the action of PTH on Na+-Pi cotransport in brush border membranes and on cAMP-dependent phosphorylation of the membrane. It is strongly suggested that PTH effects changes in Na+-dependent 32Pi transport in isolated brush border vesicles and changes in 32P phosphorylation of vesicles via a direct action on the renal cortical cell rather than as a consequence of extrarenal actions of the hormone.     

Dual effect of adrenalin on sugar transport in rat diaphragm muscle

The effect of adrenalin on the membrane transport of the non-metabolized sugar, 3-methylglucose, was studied in isolated “intact” rat hemidiaphragms and related to simultaneously occurring changes in the internal levels of Na⁺, ATP, glucose-6-P, glycerol formation and ⁴⁵Ca uptake and loss. Basal sugar transport was inhibited by low (10⁻⁸−10⁻⁵ M) concentrations of adrenalin; this was antagonized by propranolol and practolol. High concentrations (10⁻⁴−10⁻³ M) stimulated sugar transport, and this was blocked by propranolol and butoxamine and was dependent on external Ca²⁺. These results suggest interaction with two different classes of adrenergic receptors, possibly of β₁ and β₂ types. Both low and high concentrations increased Na⁺ and K⁺ gradients by a practolol-sensitive effect. Isoproterenol behaved identically but phenylephrine had only the two practolol-sensitive effects on sugar and ion transport. Insulin did not interfere with inhibition of sugar transport and decrease in internal Na⁺ but prevented stimulation of sugar transport. Under anoxia adrenalin had no effect on sugar transport but led to greater Na⁺ gain by tissue. Addition of 3.0 mM palmitate decreased inhibition of sugar transport without changing receptor specificity. ATP was decreased and lipolysis enchanged by high adrenalin but glucose-6-P was increased by the low concentration as well. Influx of ⁴⁵Ca was decreased by low and increased by high adrenalin; ⁴⁵Ca efflux was also differentially affected. The results indicate that inhibition and stimulation of sugar transport depend on different receptors and that the latter response may override the former. The data are consistent with the earlier postulated regulatory role of sarcoplasmic Ca²⁺ on sugar transport in muscle, with adrenalin affecting Ca²⁺ fluxes and distribution both directly and indirectly.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytesa. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of ¹²⁵I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37°C for 30 min or 18°C for 2 h, washing free of cell surface receptor-bound tracer at 4°C and then reincubating at 37°C. The cells preloaded at 37°C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 39}\text{min}$). When the preloaded cells were incubated in the presence of 100 μg/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 μM colchicine, 20 μM cytochalasin B, 20 μM chloroquine, 10 mM NH₄Cl, 10 μM monensin or 20 μM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoocrosomucoid were observed with cells preloaded at 18°C. These results indicate that diacytosis of ¹²⁵I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.     

Na+-dependent amino acid transport in preimplantation mouse embryos. II. Metabolic inhibitors and nature of the cation requirement.

Experiments were conducted in order to determine the energy source and nature of the cation dependency of [³H]methionine transport in preimplantation mouse embryos. The energy source of methionine transport was studied at the late four-cell and early blastocyst stages. The embryos, raised in vitro, were incubated for 1 hr in inhibitor(s) of energy metabolism and then transferred for 1 hr to medium that contained inhibitor(s) and ³H-methionine. These inhibitor studies suggest that respiration and glycolysis are needed to maintain uptake of methionine in early blastocysts. Late four-cell embryos seem to utilize respiration alone for transport.The cation dependency of methionine transport was studied at the late morula and early blastocyst stages. The kinetics of methionine uptake by early blastocysts in Na⁺-depleted media indicate a competitive type of inhibition. The uptake of methionine by early blastocysts is relatively resistant to ouabain and unaffected by K⁺-free medium. In contrast, methionine uptake by late morula-stage embryos is markedly inhibited by ouabain and K⁺-free medium in 1 hr. These results suggest that 1) Na⁺ serves to increase the affinity of methionine for the carrier in early blastocysts, 2) the cation gradients do not supply a major fraction of the energy required for methionine transport, and/or the gradients are difficult to perturb once the blastocyst has formed, and 3) putative Na⁺ pumps may be localized on the blastocoelic surface of the blastocysts.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.     

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions.     

Effect of glucocorticoids on the production of lactate dehydrogenase, malate dehydrogenase and alpha-fetoprotein by Morris hepatoma 7777 in vitro.

Non-AFP-producing Morris hepatoma 7777 were treated with glucocorticoids in order to compare the responses for AFP production and for lactate and malate dehydrogenases. Steroid hormone treatment did not affect the production of AFP. However, there was an approximate tripling of levels of both LDH and MDH (cytosolic plus mitochondrial).     

Stimulation of Ca2+ efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10^?10 M to 10^?7 M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.