Chromaffin cell calcium channel kinetics measured isotopically through fast calcium, strontium, and barium fluxes

Fast Ca2+ uptake into K+-depolarized cultured bovine adrenal chromaffin cells has been isotopically measured in a time scale of 1-10 s. Depolarized cells retained as much as 80-fold 45Ca2+ taken up by resting cells; Ca2+ was not taken up by fibroblasts or endothelial-like cells. Because Ca2+ entry was inhibited by inorganic (La3+, Co2+, Mg2+) and organic (nifedipine) Ca2+ channel antagonists and enhanced by the Ca2+ channel activator Bay-K-8644, it seems clear that Ca2+ gains access to the chromaffin cell cytosol mainly through specific voltage-dependent Ca2+ channels. Ca2+ uptake evoked by 59 mM K+ was linear during the first 5 s of stimulation and continued to rise at a much slower rate up to 60 s. The rate of Ca2+ entry became steeper as the external [Ca2+] increased; initial rates of Ca2+ uptake varied from 0.06 fmol/cells . s at 0.125 mM Ca2+ to 2.85 fmol/cell . s at 7.5 mM Ca2+. The early 90Sr2+ uptake was linear but faster than Ca2+ uptake and later on was also saturated; 133Ba2+ was taken up still at a much faster rate and was linear for the entire depolarization period (2-60 s). Increased [K+] gradually depolarized chromaffin cells; Ca2+ and Sr2+ uptakes were not apparent below 30 mM K+ but were linear for 30 to 60 mM K+. In contrast, substantial Ba2+ uptake was seen even in K+-free solutions; and in 5.9 mM K+, Ba2+ uptake was as high as Ca2+ uptake obtained in 60 mM K+. Five to ten-second pulses of 45Ca2+, 90Sr2+, or 133Ba2+ given at different times after pre-depolarization of chromaffin cells served to analyze the kinetics of inactivation of the rates of entry of each divalent cation. Inactivation of Ca2+ uptake was faster than Sr2+, and Ba2+ uptake inactivated very little. Neither voltage changes nor Ca2+ ions passing through the channels seems to cause their inactivation; however, experiments aimed to manipulate the levels of internal Ca2+ using the cell-permeable chelator Quin-2 or the ionophore A23187 strongly suggest that intracellular Ca2+ levels determine the rates of inactivation of these channels.     

Novel aspects of gonadotropin-releasing hormone action on inositol polyphosphate metabolism in cultured pituitary gonadotrophs

The hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone secretion via receptor-mediated activation of phosphoinositide hydrolysis to yield inositol phosphates and diacylglycerol. Application of anion-exchange high-performance liquid chromatography together with absorbance and radiochemical flow detection has enabled both the characterization and quantitative estimation of pituitary cell inositol phosphates and phosphoinositides. In cultured pituitary cells, GnRH caused a rapid and progressive rise in the formation of inositol 1,4,5-trisphosphate and of higher polyphosphoinositols corresponding to inositol tetrakisphosphate, pentakisphosphate, and hexakisphosphate. The inositol 1,4,5-trisphosphate formed during GnRH action was dephosphorylated predominantly via inositol 4-monophosphate rather than the expected metabolite, inositol 1-monophosphate. The catabolism of inositol 4-monophosphate, like that of inositol 1-monophosphate, was inhibited by lithium. For these reasons and because it was the major metabolite of [3H] inositol 1,4,5-trisphosphate in permeabilized gonadotrophs, inositol 4-monophosphate appears to represent a specific marker for ligand-stimulated inositol polyphosphate formation and metabolism. The marked and sustained elevations of inositol 4-monophosphate and inositol 1,4-bisphosphate in GnRH-stimulated gonadotrophs indicate that polyphosphoinositides rather than phosphatidylinositol are the preferred substrates of phospholipase C during GnRH action.     

2,3-Bisphosphoglycerate protects mitochondrial and cytosolic proteins from proteolytic inactivation

2,3-bisphosphoglycerate at physiological concentration similar to that found in many tissues protects effectively ornithine transcarbamoylase (OTC) from proteolytic inactivation by broken lysosomes. 2,3-bisphosphoglycerate protects also many other mitochondrial and cytosolic proteins, such as glutamate dehydrogenase (GDH) an glyceraldehyde-3-phosphate dehydrogenase (GAPDH), from proteolysis by broken lysosomes and other proteases. It is, thus, suggested that 2,3-bisphosphoglycerate may play an important role in the control of the degradative rates of some proteins, which may explain its high concentration in certain cells.     

Protein and lipid disturbances in rat liver microsomal membranes after bile duct ligation

An analysis of proteins, phospholipids and cholesterol from liver microsomal membranes was performed in normal and post-cholestatic rats. Bile duct ligated rats showed a progressive decrease of these membrane constituents. Minor changes in peptide analysis, a marked decrease of phosphatidylcholine and phosphatidylinositol, disappearance of phosphatidylethanolamine and sphingomyelin, and a clear increment of phosphatidylserine was observed in post-cholestatic as compared to normal group. It was concluded that extra-hepatic cholestasis produces structural changes on the liver microsomes, particularly on phospholipid profile.     

Effects of chronic allopurinol therapy on purine metabolism in Duchenne muscular dystrophy

Adenine, adenosine, inosine, hypoxanthine, xanthosine, xanthine, guanine and guanosine blood levels in 11 Duchenne muscular dystrophy patients treated with allopurinol, 10 untreated patients and 8 healthy controls, were determined by HPLC. Serum ADA, PNP and 5'-NT were also determined. Untreated patients showed lower adenine (p less than 0.001) and higher adenosine, xanthine, ADA and PNP levels (p less than 0.01) than controls. Treated patients had lower adenine and higher xanthine levels (p less than 0.001), but higher hypoxanthine, xanthosine and guanine levels (p less than 0.001), than controls, with normal ADA and PNP. The changes observed in ADA and PNP levels suggest an involvement of these enzymes in accelerated degradation of purines in Duchenne dystrophy.     

Cardiac glycosides stimulate phospholipase C activity in rat pinealocytes

Ouabain and related cardiac glycosides stimulate phospholipase C activity 5-fold in rat pinealocytes. The combined treatment of ouabain and norepinephrine, which also stimulates phospholipase C, produces an additive effect. The effects of either ouabain or norepinephrine are blocked by EGTA. However, there are notable differences. The stimulatory effect of ouabain is lost when extracellular Na+ is reduced to 20 mM and is not blocked by prazosin. In contrast, the stimulatory effect of norepinephrine is not blocked when extracellular Na+ is reduced to 20 mM but is blocked by prazosin. Ouabain appears to increase phospholipase C activity through a mechanism involving inhibition of Na+,K+-ATPase, and an accumulation of intracellular Na+ and Ca2+, not involving alpha 1-adrenoceptors. These findings raise the possibility that activation of phospholipase C might be a more general effect of cardiac glycosides.     

Apocytochrome-c competes with pre-ornithine carbamoyl transferase for transport into mitochondria

Apocytochrome c, the cytosolic precursor of cytochrome c, competes with the precursor of ornithine carbamoyltransferase (OCT) for entry into isolated rat liver mitochondria.