The spatial organization of the cytoskeleton in crayfish stretch receptor.

An electron microscopic study of the cytoskeleton of the crayfish stretch receptor was carried out. Longitudinal sections of the sensory neuron axons and dendrites showed wave-like arrays of microtubules with a period of about 5 microns. Transverse sections showed that the microtubules displayed no regularity in the arrays. In oblique sections, transverse and longitudinal views of microtubules (or shorter and longer segments of microtubules) alternated yielding a festoon-like pattern. The data obtained indicate that the cytoskeleton of the stretch receptor has a helical structure in which all the microtubules, the major cytoskeletal components, are arranged in parallel helices that are in register along the length of axons and dendrites. The helical organization of the cytoskeleton is probably responsible for the banded appearance of sensory axons and primary dendrites as seen in the polarized light. Decrease of contrast and disappearance of the banding during stretch of the receptor muscle are supposedly due to the desynchronization of the helical trajectories of the microtubules and to the decrease of the helical amplitude.     

Ultrastructure of neuroglial contacts in crayfish stretch receptor

In order to explore neuroglial relationships in a simple nervous system, we have studied the ultrastructure of the crayfish stretch receptor, which consists of only two mechanoreceptor neurons enwrapped by glial cells. The glial envelope comprises 10–30 glial layers separated by collagen sheets. The intercellular space between the neuronal and glial membranes is generally less than 10–15 nm in width. This facilitates diffusion between neurons and glia but restricts neuron communication with the environment. Microtubule bundles passing from the dendrites to the axon through the neuron body limit vesicular transport between the perikaryon and the neuronal membrane. Numerous invaginations into the neuron cytoplasm strengthen glia binding to the neuron and shorten the diffusion pathway between them. Double-membrane vesicles containing fragments of glial, but not neuronal cytoplasm, represent the captured tips of invaginations. Specific triads, viz., “flat submembrane cisterns - vesicles - mitochondria”, are presumably involved in the formation of the invaginations and double-membrane vesicles and in neuroglial exchange. The tubular lattice in the glial cytoplasm might transfer ions and metabolites between the glial layers. The integrity of the neuronal and glial membranes is impaired in some places. However, free neuroglial passage might be prevented or limited by the dense diffuse material accumulated in these regions. Thus, neuroglial exchange with cellular components might be mediated by transmembrane diffusion, especially in the invaginations and submembrane cisterns, by the formation of double-walled vesicles in which large glial masses are captured and by transfer through tubular lattices. This work was supported by RFBR (grants 05-04-48440 and 08-04-01322) and Minobrnauki RF (grant 2.1.1/6185).     

'Quantal' Ca2+ release and the control of Ca2+ entry by inositol phosphates--a possible mechanism

The release of Ca2+ from intracellular stores by sub-optimal doses of inositol trisphosphate has been shown to be dose-related ('quantal'), and a simple model is proposed here to account for this phenomenon. It is suggested that there is a regulatory Ca2(+)-binding site on, or associated with, the luminal domain of the InsP3 receptor, which allosterically controls Ca2+ efflux, and the affinity for Ca2+ of that site is modulated by InsP3 binding to the cytoplasmic domain of the receptor; a similar mechanism applied to the ryanodine receptor might also explain some aspects of Ca2(+)-induced Ca2+ release. The stimulated entry of Ca2+ into a cell which occurs upon activation of inositide-linked receptors has been variously and confusingly proposed to be regulated by InsP3, InsP4, and/or a 'capacitative' Ca2+ pool; the mechanism of InsP3 receptor action suggested here is shown to lead to a potential reconciliation of all these conflicting proposals.     

Ca2+ pools and cell growth. Evidence for sarcoendoplasmic Ca2+-ATPases 2B involvement in human prostate cancer cell growth control

The present study demonstrates for the first time that intracellular calcium-ATPases and calcium pool content are closely associated with prostate cancer LNCaP cell growth. Cell growth was modulated by changing the amount of epidermal growth factor, serum, and androgene in culture media. Using the microspectrofluorimetric method with Fura-2 and Mag Fura-2 as probes, we show that in these cells, the growth rate is correlated with intracellular calcium pool content. Indeed, an increased growth rate is correlated with an increase in the calcium pool filling state, whereas growth-inhibited cells show a reduced calcium pool load. Using Western blotting and immunocytochemistry, we show that endoplasmic reticulum calcium pump expression is closely linked to LNCaP cell growth, and are a common target of physiological stimuli that control cell growth. Moreover, we clearly demonstrate that inhibition of these pumps, using thapsigargin, inhibits LNCaP cell growth and prevents growth factor from stimulating cell proliferation. Our results thus provide evidence for the essential role of functional endoplasmic reticulum calcium pumps and calcium pool in control of prostate cancer LNCaP cell growth, raising the prospect of new targets for the treatment of prostate cancer.     

ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.     

Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle. II. Cytochemical demonstration of Ca2+-ATPase and Mg2+-ATPase

Periodical changes in Ca2+-ATPase and Mg2+-ATPase activity were observed cytochemically in the crayfish gastrolith epithelium during the molting cycle in relation to the calcium transport mechanism. The ATPase activity was demonstrated by a new one-step lead citrate method. The reaction products were mainly restricted to the matrix of type II cell mitochondria. The Ca2+-ATPase activity was intensely observed in two calcium moving stages, the small gastrolith period which indicates the beginning of gastrolith formation, and the aftermolt , when the calcified gastrolith has been dissolved in the stomach and then reabsorbed from the stomach epithelium into the newly formed soft exoskeleton through the blood. Although the intensity of reaction products of Mg2+-ATPase varied in each stage, the enzymatic activity was observed throughout all molting stages. Reaction products were observed in all mitochondria, basement membranes, apical cytoplasmic membranes, and in some lysosomes. In conclusion, periodical changes in the two types of ATPase activity were seen in the mitochondria of gastrolith epithelium during the molting cycle, but Ca2+-ATPase activity seemed to be more prominently synchronized to the calcium movement in the gastrolith epithelium than Mg2+-ATPase activity. There results provide the strong evidence that Ca2+-ATPase may act strongly in the calcium transport system of crayfish molting.     

Evidence for the existence of regulatory sites for Ca2+ on the Na+/Ca2+ carrier of cardiac mitochondria.

The Na+-induced efflux of Ca2+ catalysed by the Na+/Ca2+ carrier of cardiac mitochondria is strongly inhibited by extramitochondrial Ca2+. The nature of this inhibition was investigated as follows. (a) The apparent association of external Na+ and the Ca2+ analogue Sr2+ with substrate-binding sites (i.e. those sites involved in cation translocation) is promoted markedly by K+. The inhibition of Na+/Ca2+ exchange by external Ca2+ is affected little by K+. (b) There is a competitive relationship between the binding of external Na+ and external Ca2+ to substrate-binding sites, whereas at low concentrations (less than 4 microM) extramitochondrial Ca2+ is a partial non-competitive inhibitor with respect to external Na+. (c) This inhibiton by external Ca2+ is characterized by a maximal decrease of about 70% in the Vmax of Na+/Ca2+ exchange and by cooperative binding of external Ca2+ to sites that are half saturated by 0.7-0.8 microM free Ca2+. The binding of Ca2+ and Sr2+ to substrate-binding sites shows no co-operativity. These criteria suggest that the Na+/Ca2+ carrier may contain regulatory sites that render the carrier sensitive to changes in extramitochondrial [Ca2+] within the physiological range.     

Quantitative studies on the slowly adapting stretch receptor of the crayfish

Summary In isolated receptors the impulse frequency following step stretches had a highly significant correlation with both muscle length and tension; any deviations from linearity were in opposite directions, impulse frequency rising more quickly than linearly with length and more slowly than linearly with tension. The impulse frequency decayed according to a power function of time from application of a step increase in length. A transfer function was derived and used to predict responses to sinusoidal and constant velocity stretches. The experimental data generally agreed with predictions. The deviations that were found could be accounted for by considering quantitatively any non-linearity between frequency and length, the adaptation of the impulse frequency to constant currents, the all-or-none nature of the action potential, and the viscous forces present during dynamic stretch. The approximately linear relationship between impulse frequency and muscle length and muscle tension is discussed. Muscle tension appears to be the more direct causal agent of impulse generation. Possible physical bases for the transfer function are also considered.     

Transport and control of Ca2+ by pigeon erythrocytes. II. Evidence against a simple feedback control of cell Ca2+ and evidence for the involvement of more than one pool

Pigeon erythrocytes did not behave as expected from simple feedback mechanisms. The pool size for exchangeable cell Ca2+ was approximately proportional to the A23187-induced apparent 45Ca2+ influx ("J(in,app)") from 0.4 to 14 mumoles/min X l cell water at 184 microM external [Ca2+]. From earlier data, total cell 45Ca2+ was approximately proportional to J(in,app) from 10 to 120 mumoles/l X min. Thus there was no influx range where cell 45Ca2+ was held approximately constant. External [Ca2+] affected Ca2+ pool size independently of its effect of J(in,app). Trifluoperazine did not increase cell 45Ca2+ with or without A23187. In the presence of A23187, 45Ca2+ entered a pool early in the incubation which later became inaccessible to 45Ca2+ entry and exit. Lysolecithin addition produced an abrupt rise in cell 45Ca2+, much of which occupied a pool that quickly became inaccessible. The increased 45Ca2+ influx induced by lysolecithin dropped quickly and markedly with time. It is hard to explain inaccessible pool(s), especially in the presence of A23187 by membrane-bounded compartments. We suggest that nonexchangeable 45Ca2+ might be held by an energy-dependent binding protein(s).     

Intracellular regulation of oxygen consumption in the isolated crayfish stretch receptor neuron

Morphological correlations of the functional regulation of oxygen consumption have been studied on single isolated crayfish mechanoreceptor neurons. An enhancement of oxygen consumption is promoted by the following: (1) redistribution of mitochondria and an increase in cytochrome oxidase (CO) activity in mitochondria near the plasmatic membrane, (2) coordination of mitochondria aggregation rhythms with pO₂ rhythms in the medium external for a cell, (3) a decrease in the area of high CO activity and mitochondria and a shortening of the oxygen diffusion pathway, (4) an increase of the CO activity gradient from the neuron body periphery to its center, (5) a transfer of oxygen with the water flow during neuron body hydration and cytoplasm dilution during the transfer of a portion of the gel into sol, (6) cyclic changes in the ratio of the neuron body and hillock sizes at which there is a transfer of oxygen with the water flow into the neuron body, its mitochondrial uptake in the neuron body, and transfer of the oxygen-free water from the neuron body into the axonal hillock and further into the external medium.     

Ca2+ in quality control

Calcium (Ca²⁺) has long been known as a ubiquitous intracellular second messenger, exploited by cells to control processes as diverse as development, proliferation, learning, muscle contraction and secretion. The spatial and temporal patterns of these Ca²⁺-associated signals, as well as their amplitude, is precisely controlled to create gradients of the ion, varying considerably depending on cell type and function. Tuning of intracellular Ca²⁺ is achieved in part by the buffering role of mitochondria, whose unperturbed function is essential for maintaining cellular energy balance. Quality of mitochondria is ensured by the process of targeted autophagy or mitophagy, which depends on a molecular cascade driving the catabolic process of autophagy toward damaged or deficient organelles for elimination via the lysosomal pathway. Nonspecific and targeted autophagy are highly regulated processes fundamental to cell growth and tissue homeostasis, allowing resources to be reallocated in nutrient-deprived cells as well as being instrumental in the repair of damaged organelles or the elimination of those in excess. Given the role of Ca²⁺ signaling in many fundamental cellular processes requiring precise regulation, the involvement of Ca²⁺ in autophagy is still somewhat ill-defined, and only in the past few years has evidence emerged linking the two. This mini-review aims to summarize recent work implicating Ca²⁺ as an important regulator of autophagy, outlining a role for Ca²⁺ that may be even more critical in the regulation of targeted mitochondrial autophagy.     

Evidence for an alteration in the microsomal Ca2+ release mechanism in senescent rat parotid acinar cells

Previously, we have shown that Ca2+ mobilization following an alpha 1-adrenergic receptor stimulus is reduced in parotid acinar cells from senescent rats as a result of an altered ability of inositol 1,4,5-trisphosphate (IP3) to induce Ca2+ release from a non-mitochondrial, intracellular Ca2+ store (Ishikawa, Y., et al. Biochim. Biophys. Acta 968, 203-210). We have used this model to examine the IP3-induced Ca2+ release mechanism in these cells. 45Ca2+ efflux, after exposure to (-) epinephrine, from cells of young adult (3-6 months) rats was approx. 2-fold that observed from cells from older animals (approx. 24 months) either in the presence or absence of extracellular Ca2+. Similarly, cytosolic Ca2+ levels were greater in cells of young adult rats under these same incubation conditions. However, microsomal membrane preparations, from both age groups displayed similar IP3 binding sites (Kd approximately 90 nM, Bmax approximately 850 fmol/mg protein) and ATP-dependent Ca2+ transport ability (approx. 8 nmol/mg protein.min -1). These data suggest that there is an alteration in the IP3-induced Ca2+ release mechanism in microsomal membranes of parotid glands from senescent rats which may account for the decreased Ca2+ release seen after agonist stimulation of this tissue.     

Evidence for a Ca2+ channel within the ryanodine receptor complex from cardiac sarcoplasmic reticulum

The solubilized [3H]ryanodine receptor from cardiac sarcoplasmic reticulum was centrifuged through linear sucrose gradients. A single peak of radioactivity with apparent sedimentation coefficient of approximately 30S specifically comigrated with a high molecular weight protein of apparent relative molecular mass approximately 400,000. Incorporation of the ryanodine receptor into lipid bilayers induced single Ca2+ channel currents with conductance and kinetic behavior almost identical to that of native cardiac Ca2+ release channels. These results suggest that the cardiac ryanodine receptor comprises the Ca2+ release channel involved in excitation-contraction coupling in cardiac muscle.     

Negative control mechanism with features of adaptation controls Ca2+ release in cardiac myocytes.

The central paradox of cardiac excitation-contraction coupling is that Ca(2+)-induced Ca2+ release (CICR), an inherently self-regenerating process, is finely graded by surface membrane Ca2+ current (ICa). By using FPL64176, a novel Ca2+ channel agonist that reduces inactivation of ICa, a rapid negative control mechanism was unmasked at the Ca2+ release level in isolated rat ventricular myocytes. This mechanism terminates CICR independently of the duration of trigger ICa and before the sarcoplasmic reticulum becomes depleted of Ca2+. In its ability to be reactivated by incremental increases in trigger ICa, this mechanism differs from conventional inactivation/desensitization and is similar to the mechanism of increment detection or adaptation described for intracellular Ca2+ release channels. These results indicate that ryanodine receptor adaptation regulates Ca2+ release in cardiac muscle, accounting for or contributing to the graded nature of CICR and, additionally, permitting stores to reload at later times during Ca2+ entry.