Use of cultured cells of kidney origin to assess specific cytotoxic effects of nephrotoxins.

During drug discovery, assessment of renal safety for a compound is important for further development of a candidate drug. In this study, we describe an in vitro cell-based assay capable of discerning nephrotoxicity. Three cell types, two of kidney origin and one of liver origin, were used to examine the effects of nephrotoxins. The cell types were the porcine normal kidney tubular epithelial cell line (LLC-PK1), the primary human renal proximal tubular epithelial cells (hRPTEC) and the human liver cell line (HepG2). Cytotoxicity was measured using a luciferin/luciferase assay that measures cellular ATP levels. Four known nephrotoxins, 4-aminophenol, cisplatin, cyclosporin A and paraquat, were tested in this cell-based assay to evaluate cytotoxicity on drug exposure. Kidney-derived LLC-PK1 cells and hRPTECs were found to be sensitive to selected nephrotoxins while liver-derived HepG2 cells were insensitive. Human RPTEC cells obtained from three individual donors demonstrated highly reproducible effects on drug exposure. With respect to drug discovery efforts, integration of the cell models described here are valuable for evaluation of nephrotoxic potentials during lead selection and optimization processes.

Terrorist on trial: the context of political crime.

When political terrorists stand trial for their violent acts, the political context inevitably plays a major role. This article describes the trial of an Abu Nidal terrorist tried in federal court for skyjacking an Egyptian airliner. The defense portrayed the traumas of the Palestinian people and of the defendant at the hands of the Israelis, offering a not guilty by reason of insanity defense on the basis of posttraumatic stress disorder. Making sense to the jury of how a sane individual could carry out a violent act in which more than 50 innocent men, women, and children died was the task of the author, who served as expert for the U.S. Department of Justice. The paper describes how the subject was socialized to violence in the refugee camps, where he was inspired to be a soldier in the revolution in order to reclaim his family lands. Nationalist-separatist terrorism is particularly intractable because of the generational transmission of hatred and revenge.

MRI of acute spinal epidural hematomas.

PURPOSE: The purpose of this work was to determine the MR findings that characterize acute spinal epidural hematomas (ASEHs). METHOD: The MR findings of 17 patients with ASEH (9 cervical, 7 thoracic, and 2 lumbar) were reviewed. Fifteen of the hematomas were secondary to trauma and two were spontaneous. Correlation with CT (8 cases) and surgical findings (11 cases) was also performed. RESULTS: Imaging findings in ASEH were the following: (a) a variable signal intensity (on T1-weighted images, 10 showed isointensity to cord and 7 were slightly hyperintense; T2-weighted images showed hyperintensity with areas of hypointensity); (b) capping of epidural fat; (c) direct continuity with the adjacent osseous structures; (d) compression of epidural fat, subarachnoid sac, and spinal cord; (e) usually posterolateral location in the spinal canal. CONCLUSION: Epidural hematomas in the spinal canal are lesions capable of producing sudden spinal cord and/or cauda equina compression. MR provides characteristic findings that allow a prompt diagnosis of acute epidural hematomas.

Genocide in Bosnia: the case of Dr. Radovan Karadzic.

From 1992 to 1995 the Republic of Bosnia-Herzegovina experienced a war of genocidal proportions between the Bosnian Serbs, the Bosnian Croats, and the Bosnian Muslims. The international Criminal Tribunal for the Former Yugoslavia has indicted Dr. Radovan Karadzic--former President of the Bosnian Serb Republic, psychiatrist, and poet--as a suspected war criminal for his role in war crimes, crimes against humanity, and genocide. Karadzic remains enigmatic and poorly understood. Psychological profiling highlights in Karadzic's case the complex coalescence of the psychology of a genocide perpetrator with that of a charismatic narcissistic political leader. Such a profile may possess usefulness in forensic psychiatric investigations and legal proceedings.

[Ca2+]i inhibition of K+ channels in canine pulmonary artery. Novel mechanism for hypoxia-induced membrane depolarization.

Experiments were performed on smooth muscle cells isolated from canine pulmonary artery to identify the type of K+ channel modulated by hypoxia and examine the possible role of [Ca2+]i in hypoxic K+ channel inhibition. Whole-cell patch-clamp experiments revealed that hypoxia (induced by the O2 scavenger, sodium dithionite) reduced macroscopic K+ currents, an effect that could be prevented by strong intracellular buffering of [Ca2+]i. The inhibitory effects of hypoxia were mimicked by acute exposure of cells to caffeine and could be prevented by caffeine pretreatment, suggesting an important obligatory role of [Ca2+]i in hypoxic inhibition of K+ currents. Exposure of cells to low concentrations of 4-aminopyridine (4-AP, 1 mmol/L) prevented hypoxic inhibition of macroscopic K+ currents, whereas low concentrations of tetraethylammonium were without effect, suggesting that the target K+ channel inhibited by hypoxia is a voltage-dependent delayed rectifier K+ channel, which is inhibited by [Ca2+]i. Hypoxia failed to consistently modify the activity of large-conductance (118 picosiemens [pS] in physiological K+) Ca(2+)-activated K+ channels in inside-out membrane patches but reduced open probability of smaller-conductance (25-pS) delayed rectifier K+ channels in cell-attached membrane patches. In inside-out membrane patches, 1 mumol/L Ca2+ added to the cytoplasmic surface significantly reduced open probability of small-conductance (25-pS) 4-AP-sensitive delayed rectifier K+ channels. Whole-cell current measurements using symmetrical K+ to increase driving force for small currents active near the cell's resting membrane potential revealed the presence of a 4-AP-sensitive K+ current that activated near -65 mV and was inhibited by hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular divalent cations block smooth muscle K+ channels.

The patch-clamp technique was used to examine the sensitivity of delayed rectifier K+ channels to changes in intracellular divalent cations (Mg2+ and Ca2+). During voltage-step and ramp depolarizations, a delayed rectifier K+ current (IK(dr)) was identified in renal, pulmonary, coronary, and colonic smooth muscle cells as a low-noise outward current that activated near -40 mV, was sensitive to 4-aminopyridine (4-AP), and was insensitive to charybdotoxin. During whole-cell voltage-clamp experiments in each of the cell types, the 4-AP-sensitive IK(dr) was significantly less in cells dialyzed with 10 mM Mg2+ as compared with cells in which no Mg2+ was added to the internal dialysis solution (P < or = .05, n > or = 4). In coronary artery cells, 100 microM 2-(2-aminoethyl)pyridine (an H1 receptor agonist) or 10 microM ryanodine, agents that cause an increase in [Ca2+]i, also caused a significant reduction of the 4-AP-sensitive IK(dr) similar to that produced by Mg2+. 4-AP (5 mM) significantly depolarized single renal arterial cells that were dialyzed with Mg(2+)-free solution but not those dialyzed with 10 mM Mg2+ (P < .01, n = 4). In inside-out patches of renal arterial smooth muscle cells, with 200 nM charybdotoxin in the patch pipette to block large conductance Ca(2+)-activated K+ channels, a 59 +/- 10-picosiemen K+ channel that was sensitive to cytoplasmic Mg2+ was identified. In Mg(2+)-free solution, channel open probability was 0.028 +/- 0.012 (n = 8) and 0.095 +/- 0.011 (n = 8) at +40 and +80 mV, respectively. When the bath solution was changed to one containing 5 or 15 mM Mg2+, channel open probability was significantly reduced by 66% and 68% (+40 mV) or 93% and 96% (+80 mV), respectively. This decrease in the open probability of the delayed rectifier K+ channel resulted from a concentration- and voltage-dependent decrease in mean open time. At +40 mV, time constants for the open time distribution were significantly decreased from 5.5 +/- 0.52 to 1.2 +/- 0.14 milliseconds, whereas the closed time constant was significantly increased from 634 +/- 11.1 to 820 +/- 14.4 milliseconds (P < .01, n = 4). It is concluded that a 4-AP-sensitive delayed rectifier K+ channel in both vascular and visceral smooth muscle cells is modulated by changes in intracellular Ca2+ and Mg2+ that may alter membrane potential and the contractile state of smooth muscle.

Ionic basis for spontaneous depolarizations in isolated smooth muscle cells of canine colon.

The type of cell that serves as the pacemaker in the colon is presently unknown. This study evaluated the ionic basis of spontaneous depolarizations in circular smooth muscle cells isolated from canine colon using whole cell voltage and current clamp techniques. Increasing temperature increased the probability of observing spontaneous depolarizations, depolarized the resting membrane potential (RMP), and increased Ca2+ and K+ currents. Spontaneous depolarizations occurred as rhythmic events, in bursts, or as isolated events. Varying the holding potential from -100 to -40 mV inhibited a component of inward current thought to be necessary for spontaneous depolarizations. The Ca2+ channel blockers, nickel and nisoldipine, inhibited spontaneous depolarizations. Nickel caused a hyperpolarization, whereas nisoldipine did not affect RMP. Ouabain depolarized the RMP and inhibited spontaneous depolarizations. The K+ channel blocker, tetraethylammonium, depolarized the RMP and lengthened the duration of spontaneous depolarizations. The key finding is that single colon circular smooth muscle cells are capable of generating spontaneous depolarizations similar to those described for slow waves in intact tissues and that a temperature- and nickel-sensitive inward current is essential for spontaneous activity.

Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction.

Cellular mechanisms responsible for hypoxic pulmonary vasoconstriction were investigated in pulmonary arterial cells, isolated perfused lung, and pulmonary artery rings. Three K+ channel antagonists, Leiurus quinquestriatus venom, tetraethylammonium, and 4-aminopyridine, mimicked the effects of hypoxia in isolated lung and arterial rings by increasing pulmonary artery pressure and tension and also inhibited whole cell K+ currents in isolated pulmonary arterial cells. Reduction of oxygen tension from normoxic to hypoxic levels directly inhibited K+ currents and caused membrane depolarization in isolated canine pulmonary arterial smooth muscle cells but not in canine renal arterial smooth muscle cells. Nisoldipine or high buffering of intracellular Ca2+ concentration with [1,2-bis(2)aminophenoxy] ethane-N,N,N',N'-tetraacetic acid prevented hypoxic inhibition of K+ current, suggesting that a Ca(2+)-sensitive K+ channel may be responsible for the hypoxic response. These results indicate that K+ channel inhibition may be a key event that links hypoxia to pulmonary vasoconstriction by causing membrane depolarization and subsequent Ca2+ entry.

Stimulation of arterial 42K efflux by ATP depletion and cromakalim is antagonized by glyburide.

It has been suggested that cromakalim (BRL 34915)-induced vasorelaxation was associated with stimulation of ATP-sensitive K channels. The hyperpolarization resulting from activation of this mechanism might then inhibit voltage-dependent Ca2+ entry and subsequent contraction. The present study evaluated the similarities of 42K efflux stimulated by ATP depletion (verified by high-performance liquid chromatography) and by exposure to cromakalim (10 microM) in rabbit superior mesenteric arteries. Both depletion of intracellular ATP and exposure to cromakalim significantly stimulated 42K efflux (P less than 0.05). Glyburide (a selective inhibitor of ATP-sensitive K channels in pancreatic beta-cells) inhibited 42K efflux stimulated by ATP depletion and by cromakalim exposure. Glyburide (10 microM) had no significant effect on either basal 42K or the 42K efflux stimulated by norepinephrine and by K depolarization, which cause voltage and Ca2(+)-dependent activation of K channels. Glyburide therefore had a relatively selective effect on vascular smooth muscle. The glyburide-sensitive 42K efflux during ATP depletion and exposure to cromakalim was greatest in Ca2(+)-free solution (Mg raised to 10 mM). We conclude that in vascular smooth muscle both depletion of ATP and exposure to cromakalim stimulate 42K efflux via a glyburide-sensitive mechanism with properties similar to those of ATP-sensitive K channels observed in cardiac and pancreatic beta-cells.

Effect of cromakalim and lemakalim on slow waves and membrane currents in colonic smooth muscle.

The effects of cromakalim (BRL 34915) and its optical isomer lemakalim (BRL 38227) were investigated in intact tissue and freshly dispersed circular muscle cells from canine proximal colon. Cromakalim and lemakalim hyperpolarized resting membrane potential, shortened the duration of slow waves by abolishing the plateau phase, and decreased the frequency of slow waves. Glyburide, a K channel blocker, prevented the effect of cromakalim on slow-wave activity. The mechanisms of these alterations in slow-wave activity were studied in isolated myocytes under voltage-clamp conditions. Cromakalim and lemakalim increased the magnitude of a time-independent outward K current, but cromakalim also reduced the peak outward K current. Glyburide inhibited lemakalim stimulation of the time-independent background current. Nisoldipine also reduced the peak outward current, and in the presence of nisoldipine, cromakalim did not affect the peak outward component of current. This suggested that cromakalim may block a Ca-dependent component of the outward current. Lemakalim did not affect the peak outward current. We tested whether the effects of cromakalim on outward current might be indirect due to an effect on inward Ca current. Cromakalim, but not lemakalim, was found to inhibit L-type Ca channels; however, glyburide did not alter cromakalim inhibition of inward Ca current. We conclude that the effects of cromakalim and lemakalim on membrane potential and slow waves in colonic smooth muscle appear to result primarily from stimulation of a time-independent background K conductance. The effects of these compounds on channel activity may explain the inhibitory effect of these compounds on contractile activity.

BRL 34915 (Cromakalim) stimulation of 42K efflux from rabbit arteries is modulated by calcium.

BRL 34915 (Cromakalim) is a novel benzopyran that has anti-hypertensive actions. Contractile and 42K efflux responses to BRL 34915 were measured in blood vessels from the rabbit to determine whether Ca-dependent K-channels [K(Ca)] were involved. BRL 34915 increased 42K efflux from all blood vessels tested with an EC50 of 0.2 to 0.7 microM in the superior mesenteric artery (SMA). BRL 34915 also inhibited norepinephrine (NE)-stimulated 42K efflux (96%) and contraction (50%) in SMA with IC50 values (0.2-0.3 microM) which were similar to the EC50. Comparison of two vascular sites showed that the portal mesenteric vein required 10-fold higher BRL 34915 to achieve a 42K response equivalent to the maximal effect on SMA, yet the maximum response to NE was 4-fold greater in portal vein than SMA. The 42K efflux stimulated by BRL 34915 exhibited a significant decrement with time. Ca-removal increased the magnitude and stability of the BRL 34915 response in SMA and femoral arteries, whereas reapplication of Ca in the presence of BRL 34915 caused the 42K efflux to decrease rapidly. These effects of Ca were not altered by nisoldipine (NIS). The 42K efflux stimulated by BRL 34915 and by K-depolarization was additive when applied in combination in SMA. BRL 34915 had no effect on the NIS-inhibited 42K efflux (a Ca-dependent component) during K-depolarization. It is concluded that BRL 34915 acts on K-channels which differ significantly from the known properties of K(Ca)-channels and that BRL 34915 indirectly inhibits NE stimulation of contraction and K(Ca)-channels by hyperpolarizing the smooth muscle membrane.

Is "the right stuff" the right stuff?

In 1979 the author Tom Wolfe likened US astronauts to sacrificial gladiators characterized by competitiveness, independence, self-sufficiency, extroversion and a sense of moral righteousness and superiority--in short, 'the right stuff'. But are these the qualities needed for long-duration international missions? The author's hunch is that they are on the whole still necessary but not sufficient, but he emphasizes that systematic study of past behavioural reactions and analogous environments is needed if vital questions of interpersonal dynamics are to be answered correctly.