The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.     

Alkalinization stimulates the purified plasma-membrane Ca2+ pump by increasing its Ca2+ affinity.

The finding that negatively charged phospholipids activate the plasma-membrane (Ca2+ + Mg2+)-ATPase and that polycations counteract this stimulation suggest that negative charges in the environment of the ATPase protein could be important for its function. The aim of the present work was to investigate whether changing the charges on the ATPase protein itself by modifying the pH within the physiological range affects the activity of the purified plasma-membrane Ca2+ pump from stomach smooth muscle. Increasing the pH from 6.9 to 7.4 and using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) as a Ca2+ buffer, doubled the ATPase activity at 0.3 microM-Ca2+ in the presence of 100% phosphatidylcholine (PC) or after substituting 20% of the PC by negatively charged phospholipids PtdIns, PtdIns4P, phosphatidylserine and phosphatidic acid. This stimulatory effect was due to an increased affinity of the enzyme for Ca2+, while the Vmax. remained unaffected. In the case of PtdIns(4,5)P2, a stimulatory effect upon alkalinization was only observed at a PtdIns(4,5)P2 concentration of 10%. When a concentration of 20% was used, alkalinization decreased the Vmax. and no stimulatory effect on the ATPase at 0.3 microM-Ca2+ could be observed. Alkalinization not only stimulated the purified Ca2+ pump, but it also increased the activity of the enzyme in a plasma-membrane-enriched fraction from stomach smooth muscle by a factor of 2.06. The ionophore A23187-induced Ca2+ uptake in closed inside-out vesicles also increased by a factor of 2.54 if the pH was changed from 6.9 to 7.4. This finding indicates that the effect of pH is most likely to be exerted at the cytoplasmic site of the Ca2+ pump protein.     

Polyamines and neomycin inhibit the purified plasma-membrane Ca2+ pump by interacting with associated polyphosphoinositides.

We investigated the effect of spermine, spermidine, putrescine and neomycin on the activity of the plasma-membrane Ca2+ pump and on its stimulation by negatively charged phospholipids and calmodulin. Millimolar concentrations of spermine and to a lesser extent of spermidine decreased the ATPase activity in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2), without affecting the stimulation by phosphatidylinositol 4-phosphate (PIP). Sub-millimolar concentrations of neomycin inhibited the stimulation of the ATPase by PIP and by PIP2. Neomycin was more effective at the higher concentrations of PIP and PIP2. We discuss that these findings are compatible with the hypothesis that PIP and PIP2 bind to the ATPase and that several of these molecules have to be available to stimulate the ATPase.     

AlF4- reversibly inhibits ''P''-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.     

Interleukin-3 stimulates the tyrosine phosphorylation of the 140-kilodalton interleukin-3 receptor

Murine interleukin-3 (mIL-3) stimulates the rapid and transient tyrosine phosphorylation of a number of proteins in mIL-3-dependent B6SUtA1 cells. Two of these proteins, p68 and p140, are maximally phosphorylated at tyrosine residues within 2 min of addition of mIL-3. Because 125I-mIL-3 can be cross-linked to both 70- and 140-kDa proteins on intact B6SUtA1 cells, we investigated whether the tyrosine phosphorylated p68 and p140 were these two mIL-3 receptor proteins. Addition of antiphosphotyrosine antibodies (alpha PTyr Abs) to cell lysates from B6SUtA1 cells, to which 125I-mIL-3 had been disuccinimidyl suberate-cross-linked, resulted in the immunoprecipitation of 125I-mIL-3 complexed to both 70- and 140-kDa proteins. To determine if the observed immunoprecipitation pattern was due to the direct interaction of alpha-PTyr Abs with these two mIL-3 receptor proteins or with tyrosine-phosphorylated proteins that were associated with the receptor proteins, cell lysates were treated with 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, and boiled for 1 min. After removal of sodium dodecyl sulfate and 2-mercaptoethanol, alpha PTyr Abs immunoprecipitated 125I-mIL-3 cross-linked to only the 140-kDa protein. To confirm this finding, 32P-labeled B6SUtA1 cells were treated with biotinylated or fluoresceinated mIL-3. Addition of immobilized streptavidin or antifluorescein antibodies, respectively, to cell lysates from these cells resulted in the enrichment of only a 140-kDa tyrosine phosphorylated protein. Taken together, these results strongly suggest that only the 140-kDa receptor protein is tyrosine phosphorylated upon mIL-3 binding.     

The effects of carbon dioxide inhalation on plasma MHPG, plasma hormones respiratory rate, and behavior in the rhesus monkey

The effects of inhalation of air and 3 concentrations of carbon dioxide (CO2) on plasma levels of the norepinephrine metabolite, MHPG, plasma hormones, and behavioral activation were assessed in eight chair-adapted Rhesus monkeys (Macaca mulatta). In comparison to air, inhalation of 5%, 7.5% and 10% CO2 for 180 minutes produced significant dose-dependent increases in respiratory rate, plasma MHPG, cortisol, growth hormone and prolactin. CO2 at the 7.5% concentration produced peak changes in behavior at 15, growth hormone at 30, and cortisol and MHPG at 180 minutes without producing changes in prolactin. The lack of previously reported CO2 induced changes in MHPG, growth hormone and prolactin in humans exposed to 7.5% CO2 for only 15 minutes, may therefore relate to the relatively short duration of CO2 exposure.