Renal involvement in mitochondrial cytopathies

Mitochondrial cytopathies constitute a group of rare diseases that are characterized by their frequent multisystemic involvement, extreme variability of phenotype and complex genetics. In children, renal involvement is frequent and probably underestimated. The most frequent renal symptom is a tubular defect that, in most severe forms, corresponds to a complete De Toni-Debré-Fanconi syndrome. Incomplete proximal tubular defects and other tubular diseases have also been reported. In rare cases, patients present with chronic tubulo-interstitial nephritis or cystic renal diseases. Finally, a group of patients develop primarily a glomerular disease. These patients correspond to sporadic case reports or can be classified into two major defects, namely 3243 A>G tRNALEU mutations and coenzyme Q10 biosynthesis defects. The latter group is particularly important because it represents the only treatable renal mitochondrial defect. In this Educational Review, the principal characteristics of these diseases and the main diagnostic approaches are summarized

H⁺-dependent calcium uptake into an IP₃-sensitive calcium pool from rat parotid gland

In permeabilized parotid cells and in isolated membrane vesicles from parotid endoplasmic reticulum (ER), Mg-ATP-dependent Ca2+ uptake was measured using a Ca2+-specific macroelectrode and 45Ca2+, respectively. Mg-ATP-dependent Ca2+ uptake was inhibited by vanadate (2 x 10-3 mol/l) by approximately 45% in permeabilized cells and by approximately 70% in membrane vesicles from ER during the initial 10 min. After this lag phase, Ca2+ uptake increased and low steady-state free [Ca2+] of approximately 3 x 10-7 mol/l was still reached in presence of vanadate within 30-40 min. Subsequent addition of inositol 1,4,5-trisphosphate (IP3) caused a similar Ca2+ release compared with control. This indicates that in presence of vanadate an IP3-sensitive Ca2+ pool was filled. However, when protonophores, such as nigericin or carbonyl cyanide-m-chlorophenylhydrazone, were added in addition to vanadate, this low steady-state free [Ca2+] was not reached. 45Ca2+ uptake was reduced by approximately 70% within 60 min, and IP3 did not cause 45Ca2+ release when given subsequently, indicating that filling of an IP3-sensitive Ca2+ pool was prevented. Mg-ATP-driven H+ uptake into ER vesicles, as estimated with acridine orange, was abolished by protonophores and by the H+-ATPase blockers N-ethylmaleimide and Dio 9 but was unaltered by vanadate. Preincubation of ER vesicles in a medium without Ca2+, but with vanadate and with Mg-ATP to generate an H+ gradient, allowed demonstration of 45Ca2+ uptake from a medium that did not contain ATP. The cation sequence in absence of vanadate for support of Mg-ATP-dependent 45Ca2+uptake was K++ greater than Na+ greater than Li+ = choline+ and, in presence of vanadate, was choline+ greater than Li+ = Na+ greater than K+. A preformed H+ gradient dissipated more rapidly in presence of K+ compared with choline+, probably due to an intrinsic K+ permeability of ER membrane. Our data indicate that both a Ca2+ and a H+ pump are located in a compartment of ER that is also sensitive to IP3. Ca2+ uptake is coupled to an H+ gradient that is generated by the H+ pump and most likely occurs via Mg-ATP-driven Ca2+-H+ countertransport but to some extent can also operate in absence of ATP at the expense of the H+ gradient

Large-scale purification of calf pancreatic zymogen granule membranes

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl− conductance, a Cl−/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5–10 mg membrane protein from ∼1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl− channels and regulatory proteins, since they contain fewer proteins than plasma membranes

Modulation of intracellular free Ca²⁺ concentration by IP₃-sensitive and IP₃-insensitive nonmitochondrial Ca²⁺ pools

Intracellular Ca2+ pools play an important role in the adjustment of cytosolic free Ca2+ concentrations. This review summarizes the recent knowledge on receptor-mediated Ca2+ release and Ca2+ uptake mechanisms in Ca2+ stores of exocrine cells taking the exocrine pancreas and the parotid gland as an example. The intracellular mediator for agonist-induced Ca2+ release is inositol 1,4,5-trisphosphate (IP3) which acts by opening Ca2+ channels from the endoplasmic reticulum or a more specialized organelle called ‘calciosome’. This Ca2+ release is the major event to increase cytosolic free Ca2+ concentrations of exocrine glands from a resting level of ∼ 10−7 mol/l to ∼ 10−6 mol/l. Subsequently also Ca2+ influx from the extracellular fluid into the cell is increased which involves the action of inositol 1,3,4,5-tetrakisphosphate (IP4). Intracellular nonmitochondrial Ca2+ reuptake occurs into IP3-sensitive (IsCaP) as well as into IP3-Insensitive Ca2+ pools (IisCaP). While Ca2+ uptake into the IisCaP is mediated by a vanadate-sensitive Ca2+ pump, Ca2+ uptake into the IsCaP is mediated by a exchanger at the expense of an H+ gradient which is established by a vacuolar type H+ pump present in the same Ca2+ pool. During stimulation both Ca2+ pools, IsCaP and IisCaP, are probably connected, the nature of which has not yet been clarified. It is suggested that GTP and/or IP4 control Ca2+ conveyance between intracellular Ca2+ pools by forming Ca2+-carrying junctions between membranes. Other models propose that Ca2+, which is released by IP3, induces Ca2+ release from another Ca2+ pool. Taking into account that H+ transport is present in IP3-sensitive Ca2+ pools the possibility of pH-regulated Ca2+ channels in the IisCaP, located in close neighbourhood to the IsCaP, is also considered

Sulfonylurea-mediated stimulation of insulin exocytosis via an ATP-sensitive K+ channel-independent action.

Several reports indicate that hypoglycemic sulfonylureas augment Ca(2+)-dependent insulin secretion via mechanisms other than inhibition of the ATP-sensitive K(+) channel. The effect involves a 65-kd protein in the granule membrane and culminates in intragranular acidification. Lowering of granule pH is necessary for the insulin granule to gain release competence. Proton pumping into the granule is driven by a v-type H(+)-ATPase, but requires simultaneous Cl(-) uptake into the granule via metabolically regulated ClC-3 Cl(-) channels to maintain electroneutrality. Here we discuss the possibility that modulation of granule ClC-3 channels represents the mechanism whereby sulfonylureas directly potentiate the beta-cell exocytotic machinery