ABSTRACT
1. The Ca(2+)-sensitive fluorescent indicator rhod-2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura-2 was used in combination with rhod-2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. 2. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half-time (t1/2) to peak for the increase in [Ca2+]m was considerably longer than the t1/2 to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. 3. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t1/2 to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. 4. Using a wide-field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. 5. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. 6. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR.
Subject(s)
Calcium/metabolism , Homeostasis/physiology , Mitochondria/metabolism , Muscle, Smooth/metabolism , Animals , Bufo marinus , Caffeine/pharmacology , Cells, Cultured , Cytoplasm/metabolism , Electric Stimulation , Fluorescent Dyes/pharmacokinetics , Heterocyclic Compounds, 3-Ring , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Membrane Potentials/drug effects , Microscopy, Video , Muscle, Smooth/cytology , Patch-Clamp Techniques , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , StomachABSTRACT
BACKGROUND: To determine if the Ki-67 (MIB-1 clone) proliferative index (PI) has prognostic potential in patients with recurrent astroglial neoplasms. METHODS: We conducted a retrospective review of 27 patients whose initial and recurrent specimens were available. Histopathology was determined according to the World Health Organization classification. Proliferation index was calculated on formalin-fixed tissue using the Ki-67 (MIB-1 clone) antibody. Morphometric data were analyzed in conjunction with clinical data and Cox Proportionate Hazards Analysis, Spearman's correlation coefficient and Mann-Whitney Test. RESULTS: Initial histopathology included 14 glioblastoma multiforme, 7 anaplastic astrocytoma, 3 oligoastrocytoma, and 3 astrocytoma. Recurrent specimens showed changes consistent with treatment. While univariate analysis shows initial histology correlated with survival (p<0.036), PI did not correlate with survival after either initial (p = 0.86) or recurrent (p = 0.46) surgery for any tumor type. PI difference between specimens also did not correlate with survival (p = 0.91). Initial PI did not correlate with recurrent PI either (p = 0.43). CONCLUSIONS: Ki-67 PI does not confer additional prognostic information for patients with recurrent astroglial neoplasms. One possible explanation for this observation is that treatment may alter the PI independent of its effect on tumor growth.
