Aquaporins in spinal cord injury: the janus face of aquaporin 4.

Although malfunction of spinal cord water channels (aquaporins, AQP) likely contributes to severe disturbances in ion/water homeostasis after spinal cord injury (SCI), their roles are still poorly understood. Here we report and discuss the potential significance of changes in the AQP4 expression in human SCI that generates glial fibrillary acidic protein (GFAP)-labeled astrocytes devoid of AQP4, and GFAP-labeled astroglia that overexpress AQP4. We used a rat model of contusion SCI to study observed changes in human SCI. AQP4-negative astrocytes are likely generated during the process of SCI-induced replacement of lost astrocytes, but their origin and role in SCI remains to be investigated. We found that AQP4-overexpression is likely triggered by hypoxia. Our transcriptional profiling of injured rat cords suggests that elevated AQP4-mediated water influx accompanies increased uptake of chloride and potassium ions which represents a protective astrocytic reaction to hypoxia. However, unbalanced water intake also results in astrocytic swelling that can contribute to motor impairment, but likely only in milder injuries. In severe rat SCI, a low abundance of AQP4-overexpressing astrocytes was found during the motor recovery phase. Our results suggest that severe rat contusion SCI is a better model to analyze AQP4 functions after SCI. We found that AQP4 increases in the chronic post-injury phase are associated with the development of pain-like behavior in SCI rats, while possible mechanisms underlying pain development may involve astrocytic swelling-induced glutamate release. In contrast, the formation and size of fluid-filled cavities occurring later after SCI does not appear to be affected by the extent of increased AQP4 levels. Therefore, the effect of therapeutic interventions targeting AQP4 will depend not only on the time interval after SCI or animal models, but also on the balance between protective role of increased AQP4 in hypoxia and deleterious effects of ongoing astrocytic swelling.

Dependence of pituitary hormone secretion on the pattern of spontaneous voltage-gated calcium influx. Cell type-specific action potential secretion coupling.

In excitable cells, voltage-gated calcium influx provides an effective mechanism for the activation of exocytosis. In this study, we demonstrate that although rat anterior pituitary lactotrophs, somatotrophs, and gonadotrophs exhibited spontaneous and extracellular calcium-dependent electrical activity, voltage-gated calcium influx triggered secretion only in lactotrophs and somatotrophs. The lack of action potential-driven secretion in gonadotrophs was not due to the proportion of spontaneously firing cells or spike frequency. Gonadotrophs exhibited calcium signals during prolonged depolarization comparable with signals observed in somatotrophs and lactotrophs. The secretory vesicles in all three cell types also had a similar sensitivity to voltage-gated calcium influx. However, the pattern of action potential calcium influx differed among three cell types. Spontaneous activity in gonadotrophs was characterized by high amplitude, sharp spikes that had a limited capacity to promote calcium influx, whereas lactotrophs and somatotrophs fired plateau-bursting action potentials that generated high amplitude calcium signals. Furthermore, a shift in the pattern of firing from sharp spikes to plateau-like spikes in gonadotrophs triggered luteinizing hormone secretion. These results indicate that the cell type-specific action potential secretion coupling in pituitary cells is determined by the capacity of their plasma membrane oscillator to generate threshold calcium signals.

Differential expression of ionic channels in rat anterior pituitary cells.

Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+ -sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+ -activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.

Characterization of purinergic receptors and receptor-channels expressed in anterior pituitary cells.

Purinergic G protein-coupled receptors (P2YR) and ion-conducting receptor-channels (P2XR) are present in the pituitary. However, their identification, expression within pituitary cell subpopulations, and the ability to elevate intracellular Ca2+ concentration ([Ca2+]i) in response to ATP stimulation were incompletely characterized. Here we show that mixed populations of rat anterior pituitary cells express messenger RNA transcripts for P2Y2R, P2X2aR, P2X2bR, P2X3R, P2X4R, and P2X7R. The transcripts and functional P2Y2R were identified in lactotrophs and GH3 cells, but not in somatotrophs and gonadotrophs, and their activation by ATP led to an extracellular Ca2+-independent rise in [Ca2+]i in about 40% of cells tested. Lactotrophs and GH3 cells, but not somatotrophs, also express transcripts for P2X7R, P2X3R, and P2X4R. Functional P2X7R were identified in 74% of lactotrophs, whereas 50% of these cells expressed P2X3R and 33% expressed P2X4R. Coexpression of these receptor subtypes in single lactotrophs was frequently observed. Purified somatotrophs expressed transcripts for P2X2aR and P2X2bR, and functional receptors were identified in somatotrophs and gonadotrophs, but not in lactotrophs. Consistent with the cell-specific expression of transcripts for P2X2R and P2X3R, the expression of their functional heteromers was not evident in pituitary cells. Receptors differed in their capacities to elevate and sustain Ca2+ influx-dependent rise in [Ca2+]i during the prolonged ATP stimulation. These results indicate that the purinergic system of anterior pituitary is extremely complex and provides an effective mechanism for generating a cell- and receptor-specific Ca2+ signaling pattern in response to a common agonist.

Characterization of a plasma membrane calcium oscillator in rat pituitary somatotrophs.

In excitable cells, oscillations in intracellular free calcium concentrations ([Ca(2+)](i)) can arise from action-potential-driven Ca(2+) influx, and such signals can have either a localized or global form, depending on the coupling of voltage-gated Ca(2+) influx to intracellular Ca(2+) release pathway. Here we show that rat pituitary somatotrophs generate spontaneous [Ca(2+)](i) oscillations, which rise from fluctuations in the influx of external Ca(2+) and propagate within the cytoplasm and nucleus. The addition of caffeine and ryanodine, modulators of ryanodine-receptor channels, and the depletion of intracellular Ca(2+) stores by thapsigargin and ionomycin did not affect the global nature of spontaneous [Ca(2+)](i) signals. Bay K 8644, an L-type Ca(2+) channel agonist, initiated [Ca(2+)](i) signaling in quiescent cells, increased the amplitude of [Ca(2+)](i) spikes in spontaneously active cells, and stimulated growth hormone secretion in perifused pituitary cells. Nifedipine, a blocker of L-type Ca(2+) channels, decreased the amplitude of spikes and basal growth hormone secretion, whereas Ni(2+), a blocker of T-type Ca(2+) channels, abolished spontaneous [Ca(2+)](i) oscillations. Spiking was also abolished by the removal of extracellular Na(+) and by the addition of 10 mM Ca(2+), Mg(2+), or Sr(2+), the blockers of cyclic nucleotide-gated channels. Reverse transcriptase-polymerase chain reaction and Southern blot analyses indicated the expression of mRNAs for these channels in mixed pituitary cells and purified somatotrophs. Growth hormone-releasing hormone, an agonist that stimulated cAMP and cGMP productions in a dose-dependent manner, initiated spiking in quiescent cells and increased the frequency of spiking in spontaneously active cells. These results indicate that in somatotrophs a cyclic nucleotide-controlled plasma membrane Ca(2+) oscillator is capable of generating global Ca(2+) signals spontaneously and in response to agonist stimulation. The Ca(2+)-signaling activity of this oscillator is dependent on voltage-gated Ca(2+) influx but not on Ca(2+) release from intracellular stores.

Expression of Ca(2+)-mobilizing endothelin(A) receptors and their role in the control of Ca(2+) influx and growth hormone secretion in pituitary somatotrophs.

The expression and coupling of endothelin (ET) receptors were studied in rat pituitary somatotrophs. These cells exhibited periods of spontaneous action potential firing that generated high-amplitude fluctuations in cytosolic calcium concentration ([Ca(2+)](i)). The message and the specific binding sites for ET(A), but not ET(B), receptors were found in mixed pituitary cells and in highly purified somatotrophs. The activation of these receptors by ET-1 led to an increase in inositol 1,4,5-trisphosphate production and the associated rise in [Ca(2+)](i) and growth hormone (GH) secretion. The Ca(2+)-mobilizing action of ET-1 lasted for 2-3 min and was followed by an inhibition of action potential-driven Ca(2+) influx and GH secretion to below the basal levels. As in somatostatin-treated cells, the ET-1-induced inhibition of spontaneous electrical activity and Ca(2+) influx was accompanied by the inhibition of adenylyl cyclase and by the stimulation of inward rectifier potassium current. In contrast to somatostatin, ET-1 did not inhibit voltage-gated Ca(2+) channels. During prolonged agonist stimulation a gradual recovery of Ca(2+) influx and GH secretion occurred. In somatotrophs treated with pertussis toxin overnight, the ET-1-induced Ca(2+)-mobilizing phase was preserved, but it was followed immediately by facilitated Ca(2+) influx and GH secretion. Both somatostatin- and ET-1-induced inhibitions of adenylyl cyclase activity were abolished in pertussis toxin-treated cells. These results indicate that the transient cross-coupling of Ca(2+)-mobilizing ET(A) receptors to the G(i)/G(o) pathway in somatotrophs provides an effective mechanism to change the rhythm of [Ca(2+)](i) signaling and GH secretion during continuous agonist stimulation.

In vitro and in vivo thermal activation of steroid-receptor complexes from rats and ground squirrels (Spermophilus citellus).

Using 3H-labelled triamcinolone acetonide (3HTA, synthetic steroid hormone), it was shown that the in vitro time course kinetics of thermal activation of 3HTA-receptor complexes exhibited the same temperature dependence in liver cytosols prepared from hibernating ground squirrels (Spermophilus citellus) as in cytosols from the rat. When 3HTA was injected in vivo to animals hibernating with a body temperature of 3 degrees C, the activation and nuclear uptake of the in vivo formed steroid-receptor complexes proceeded at a slow rate, comparable to the one predicted by in vitro studies. In the hibernator, the results are not indicative of adaptive modifications at the level of thermal activation, but prove that steroid action does proceed at a temperature incompatible with hypothermic survival in the nonhibernator.