Pflügers Archiv

We investigated the mechanism by which lytic granules extracted from cytotoxic T lymphocytes (CTL) damage guinea pig ventricular myocytes in order to dertermine whether their actions can be related to the overall immunological rejection of the transplanted heart. Granule-induced myocyte morphological changes and final destruction were preceded by shortening of action potential duration (APD) and reductions of the resting potential and the action potential amplitude. APD shortening was probably caused by a granule-induced increase in outward current (most likely non-specific). Ryanodine, which blocks Ca2+ release from the sacroplasmic reticulum, did not interfere with the morphological and electrophysiological effects of lytic granules. Fura-2 imaging indicated that [Ca2+]i initially increased about 2-fold from 90.0±11.5 nM, while cell length decreased less than 5% from a mean value of 99.0±9.0 μm. A further increase in [Ca2+]i (>10 fold) was associated with progressive contracture and destruction, suggesting that the structural damage inflicted by lytic granules is caused by [Ca2+]i overload. The results indicate that the cytocidal action of CTL-derived lytic granules may be involved in immunologically induced damage, even to the extent of rejection of the transplanted heart.

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.

The structural inhomogeneity of the myocardial capillary bed is simulated by microcirculatory units (MCU's) in a diffusion model. This simulation is based on MCU's in which the arrangement of the capillary ends (concurrent structure, partial and total countercurrent structure, helical structure) as well as the structure and supply parameters are varied. The variation of these parameters is based on own measurements of the intracapillary HbO2 saturation as well as on the following parameters from the literature: frequency distribution of capillary distance and capillary radius, mean capillary length or capillary section length respectively, arterial and mean venousPO2, mean coronary blood flow, mean O2 consumption and diffusion conductivity. The analysis of O2 supply of the normoxic rat heart shows that an O2 diffusion shunt is obligatory except for MCU's with an extremely large capillary distance or with a concurrent capillary structure. Therefore the minimal tissuePO2 lies at the level of the capillary venousPO2 of a MCU. The maximum of the totalPO2 frequency distribution in the normoxic rat myocardium lies at 25±5 mm Hg, i.e. above the mean venousPO2 (20 mm Hg). TissuePO2 values between 0 and 5 mm Hg amount to 0.5%, i.e. they are extremely rare. TissuePO2 values of 0–1 mm Hg represent less than 0.2%.

 Recently, the cell-volume-regulated serine-threonine protein kinase h-sgk was cloned from a human hepatoma cell line. The sgk gene was shown to be induced by cell shrinkage in many different mammalian cell lines. In this study, two highly conserved serine-threonine protein kinases, sgk-1 and sgk-2, were cloned from rectal gland tissue of the spiny dogfish (Squalus acanthias). Both kinases showed a distinct pattern of tissue specificity, with high expression levels in kidney, intestine, liver and heart. In rectal gland slices sgk-1 transcription was induced by exposure to hypertonic solution, reduction of the extracellular urea concentration, and addition of the secretagogues vasoactive intestinal polypeptide (VIP) and carbachol. The shark sgk-1 serine-threonine protein kinase may therefore provide a link between cell volume, Cl–secretion and protein phosphorylation state in shark rectal gland cells.

Although net Ca2+ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca2+ entry step across the luminal membrane is mediated by Ca2+ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca2+ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca2+ ([Ca2+]i) with fura-2. The resting [Ca2+]i in this segment was 49.8±4.5 nmol/l. Neither Na+ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca2+]i, indicating that a Na+/Ca2+ exchanger in the basolateral membrane may not contribute in any major way to [Ca2+]i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na+/Ca2+ exchanger has been reported. In this segment, Na+ removal from the bath increased cell Ca2+ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca2+]i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca2+]i. When the bath pH was made alkaline, [Ca2+]i was also increased. This response was abolished when Ca2+ was eliminated from the bath, indicating that the Ca2+ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca2+]i induced by an alkaline bath was inhibited by increased the bath K+ from 5 nmol/l to 50 mmol/l, suggesting that the Ca2+ entry system is voltage-dependent. However, the pH-dependent [Ca2+]i increase was unaffected by 0.1–10 μmol/l nicardipine in the bath. We conclude that Ca2+ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca2+ rather than a Na+/Ca2+ exchanger secondarily linked to a Na+, K+ pump.