Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons

We have studied the Ca(2+) leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca(2+) in the endoplasmic reticulum ([Ca(2+)](ER)). Cytosolic Ca(2+) ([Ca(2+)](C)) was clamped to the resting level by a BAPTA-Ca(2+) mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca(2+)](ER), and the rate of Ca(2+) release generated by 10 pm cholecystokinin is at least 3x as fast as the basal Ca(2+) leak revealed by inhibition of the endoplasmic reticulum Ca(2+)-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca(2+)](ER), with an EC(50) of 0.98 +/- 0.06 microm. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP(3)) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH(2) cyclic ADP-ribose (20 microm) inhibits the action of cholecystokinin, but not of acetylcholine(.) The basal Ca(2+) leak from the endoplasmic reticulum is not blocked by antagonists of the IP(3) receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with puromycin (0.1-1 mm) to remove nascent polypeptides from ribosomes increases Ca(2+) leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca(2+) pumps and of the receptors for IP(3) or ryanodine.     

How to measure Ca2+ in cellular organelles?

The review will aim to briefly summarise information on calcium measurements in cellular organelles with emphases on studies conducted in live cells using optical probes. When appropriate we will try to compare the effectiveness of different indicators for intraorganellar calcium measurements. We will consider calcium measurements in endoplasmic reticulum, Golgi apparatus, endosomes/lysosomes, nucleoplasm, nuclear envelope, mitochondria and secretory granules.     

Role of mitochondria in Ca(2+) homeostasis of mouse pancreatic acinar cells

The effects of mitochondrial Ca(2+) uptake on cytosolic Ca(2+) concentration ([Ca(2+)](c)) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca(2+) indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Delta[Ca(2+)](c)=46 +/- 6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca(2+) transient, produced a larger cytosolic calcium elevation (Delta[Ca(2+)](c)=84 +/- 6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca(2+)](m)) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca(2+) uniporter), the decay time-constant of the Tg/ACh-induced Ca(2+) response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca(2+) indicator rhod-2 revealed large increases in [Ca(2+)](m) in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10nM ACh elicited large global increases in [Ca(2+)](c), compared to control cells in which ACh-induced Ca(2+) signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca(2+) homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.     

The distribution of the endoplasmic reticulum in living pancreatic acinar cells

Studies on pancreatic acinar cells provided the original evidence for the Ca(2+) releasing action of inositol 1,4,5-trisphosphate (IP(3)). Ironically, this system has presented problems for the general theory that IP(3) acts primarily on the endoplasmic reticulum (ER), because the IP(3)-elicited Ca(2+) release occurs in the apical pole, which is dominated by zymogen granules (ZGs) and apparently contains very little ER. Using confocal and two-photon microscopy and a number of different ER-specific fluorescent probes, we have now investigated in detail the distribution of the ER in living pancreatic acinar cells. It turns out that although the bulk of the ER, as expected, is clearly located in the baso-lateral part of the cell, there is significant invasion of ER into the granular pole and each ZG is in fact surrounded by strands of ER. This structural evidence from living cells, in conjunction with recent functional studies demonstrating the high Ca(2+) mobility in the ER lumen, provides the framework for a coherent and internally consistent theory for cytosolic Ca(2+) signal generation in the apical secretory pole, in which the primary Ca(2+) release occurs from ER extensions in the granular pole supplied with Ca(2+) from the main store at the base of the cell by the tunnel function of the ER.     

Activation of morphogenesis and proliferation by low specificity chemical effectors

Activation of highly specific biochemical processes by simple chemical agents is demonstrated for morphogenesis (anlage and development of female gametophyte in cereal) and mitosis (in cell cultures and animal and plant tissues). The effects of these agents are tissue-specific. Structure--activity relationship is analyzed in this group of compounds. Thus, the phenomenon reveals the exact pathways of the influence of allelopathic and anthropogenic chemical agents on evolution of plant biocenoses.     

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.     

Romanowsky staining,the Romanowsky effect and thoughts on the question of scientific priority

I give an historical account and analysis of the scientific priority of the discovery of the polychrome staining of microscopic biological preparations provided by mixtures of eosin plus methylene blue and its derivatives, especially azure B. I maintain that both the formal priority for the discovery of the polychrome staining phenomenon and credit for initiating the development of a technique of polychrome staining properly belong to D. L. Romanowsky. His scientific work demonstrated the possibility of using a simple technique to stain hematological preparations selectively to give good contrast, high resolution and the ability to identify malaria parasites. Romanowsky’s approach constituted the starting point for the development of a family of polychrome stains for microscopic investigation of hematological preparations by a number of his contemporaries.