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Background: Blastomycosis is caused by a dimorphic fungus that can be difficult to diagnose in certain situations. The disease is sometimes serious and can be deadly. Diagnosis by fungal serology and urinary antigens is not easy to establish and unreliable. Culture is also time-consuming and is not easy to perform. Thus, documentation of such an organism on cytology offers a quick and cost-effective alternative. This report describes for the first time identification of the ‘negative image’ of Blastomyces budding yeast. Case: A 79-year-old man presented with a left lung nodule associated with mediastinal and hilar lymphadenopathy. Fine needle aspiration was performed, and a ‘negative image’ of a yeast with wide base budding was noted on Diff-Quik (DQ)-stained smears. Blastomyces species were confirmed with periodic acid-Schiff fungal stain. Additionally, the fungal capsule contained focally polarizable material on Congo red stain and lacked mucin with mucicarmine stain. Conclusion:Blastomyces yeast forms can be easily identified with DQ staining by their ‘negative image’. This feature can be utilized as a quick and cost-effective cytological characteristic to further triage these specimens for confirmation. The information can be of great value to clinicians in making appropriate clinical decisions.

Myogenic stem cells and transmyocardial laser revascularization (TMR) have been used independently to treat patients with coronary heart disease. We assessed the hypothesis that implantation of autologous myogenic stem cells dur- ing TMR will augment ventricular function by new muscle formation and will improve perfusion by neovascularization. Thirty two mini-swine were subjected to myocardial ischemia by applying an ameroid constrictor on the left anterior de- scending coronary artery. Pigs were randomly assigned into four equal size groups of Ischemia, Ischemia+TMR, Ische- mia+Cell and Ischemia+TMR+Cell. Myocardial blood flow was estimated using colored microspheres, 12 lead ECG was recorded, and ventricular function was determined by the hemodynamic system coupled with sonomicrometry. Three weeks after ameroid constrictor application, ECG changes indicated myocardial infarction of left anterior and apical areas in all pigs. Significant decreases (P

Cell swelling may contribute to acute cell injury subsequent to ischemia/reperfusion. The potential role of mitochondrial uncoupling and the resultant mitochondrial swelling, due to opening of the mitochondrial permeability transition pore (MPTP), were examined in an in vitro ischemically pelleted isolated rabbit cardiomyocyte model using the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) to uncouple mitochondria. Cyclosporin A (CsA) was employed to inhibit MPTP opening. Cell volume was determined by a cell-flotation, density-gradient assay, using bromododecane. Cell viability, subsequent to an osmotic stress, was determined by trypan blue permeability. Ischemic preconditioning (IPC) facilitated volume regulation following an osmotic stress. Ischemic-cell swelling was reduced by IPC. IPC protected ischemically pelleted cells, but CsA had no significant effects on injury or IPC protection. CCCP ischemia accelerated rates of ischemic contracture and injury, and abolished IPC protection. IPC protection was restored by CsA. In CCCP-ischemic-uncoupled cells, subjected to a reduced (170 mOsm) osmotic stress, CsA and IPC afforded independent and additive protection. Chelerythrine and 5-hydroxydecanoate (5-HD) blocked IPC, but did not reduce CsA protection. Electron microscopy confirmed that CCCP ischemia induced mitochondrial matrix swelling that was reduced by CsA. Cardioprotection by IPC and CsA was accompanied by proportional reductions in cell swelling. Morphometric analysis of the electron photomicrographs demonstrated that the mitochondrial volume fractions were significantly reduced in the CsA/CCCP (29.8 +/- 2.3%, P < 0.004) and IPC/CsA/CCCP (31.5 +/- 1.7%, P < 0.0008) groups as compared to the CCCP-ischemic group (40.5 +/- 1.7%) The IPC/CCCP group (39.5 +/- 4.2%) was not significantly different from the CCCP-ischemic group. NIM 811, a CsA analogue MPTP blocker with no calcineurin inhibitory activity, afforded protection similar to CsA. The results suggest that CsA protection may, in part, be mediated by reduction of mitochondrial swelling.

A loss of sarcolemmal dystrophin was observed by immuno-fluorescence studies in rabbit hearts subjected to in situ myocardial ischemia and by immuno-blotting of the Triton soluble membrane fraction of isolated rabbit cardiomyocytes subjected to in vitro ischemia. This ischemic loss of dystrophin was a specific event in that no ischemic loss of sarcolemmal α-sarcoglycan, γ-sarcoglycan, αDG, or βDG was observed. The maintenance of sarcolemmal βDG (43 Kd) during ischemia was interesting in that dystrophin binds to the C-terminus of βDG. However, during late in vitro ischemia, a 30 Kd band was observed that was immuno-reactive for βDG. Additionally, this 30 Kd-βDG band was observed in rabbit myocardium subjected to autolysis. Finally, the 30 Kd-βDG was observed in the purified sarcolemmal fraction of rabbit cardiomyocytes subjected to a prolonged period of in vitro ischemia, confirming the sarcolemmal localization of this band. The potential patho-physiologic significance of this band was indicated by the appearance of this band at 120–180 min of in vitro ischemia, directly correlating with the onset of irreversible injury, as manifested by osmotic fragility. Additionally the appearance of this band was significantly reduced by the endogenous cardioprotective mechanism, in vitro ischemic preconditioning, which delays the onset of osmotic fragility. In addition to dystrophin, βDG binds caveolin-3 and Grb-2 at its C-terminus. The presence of Grb-2 and caveolin-3 in the membrane fractions of oxygenated and ischemic cardiomyocytes was determined by Western blotting. An increase in the level of membrane Grb-2 and caveolin-3 was observed following ischemic preconditioning as compared to control cells. The formation of this 30 Kd-βDG degradation product is potentially related to the transition from the reversible to the irreversible phase of myocardial ischemic cell injury and a decrease in 30 Kd-βDG might mediate the cardioprotection provided by ischemic preconditioning.

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ~60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 microM calyculin A, or 1 microM N(6)-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl(-) channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.

The report by Cave et al. [21] was a landmark paper which demonstrated that functional recovery protection by ischemic preconditioning (IPC) is not mediated by adenosine in the globally ischemic rat heart. This report raised several issues in the field of preconditioning. (1) It was enigmatic in that recent studies had demonstrated a predominant role of adenosine-receptors in preconditioning in the rabbit and dog so that the inability of adenosine to precondition in the rat implied that species differences might exist in the receptor pathways that lead to initiation of preconditioning. (2) Since rat cardiomyocyte metabolism preferentially relies upon exogenous glucose to support glycolysis, whereas the rabbit, dog, pig and man are more dependent upon endogenous glycogen, this report opened the possibility that species-dependent metabolic differences could also be involved in the mechanisms of IPC. (3) Since adenosine did not appear to precondition the rat, emphasis was placed on multiple G-protein coupled receptors that can induce preconditioning, e.g., α1-adrenergic-receptors in the rat [1], and on focused research on postreceptor, downstream events in the search for the ultimate mediators of protection. (4) Finally, the manuscript focused debate on the relationships between the mechanisms of preconditioning as assessed by functional recovery (stunning), reductions in arrythmias or ischemic cell death (infarction). Aortic flow during reperfusion is a sensitive end-point commonly used to assess postischemic functional recovery. Postischemic functional recovery is influenced by multiple parameters including: (1) degree of stunning during ischemia; (2) rate of anaerobic glycolysis; (3) ability to recover oxidative respiration, intracellular pH and ionic equilibrium; and (4) degree of diastolic contracture and vascular integrity at onset of reperfusion. Increased diastolic pressures subsequent to ischemic contracture reduce subendocardial perfusion such that zones of no-reflow may occur upon reperfusion. These subendocardial zones of no-reflow are a perfused heart artifact due …

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit, Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia, SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents, Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylated. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation, SB203580 accelerated ischemic hsp27 dephosphorylation and injury, IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (> 60-90 min) ischemia.

Calcium-tolerant rabbit cardiomyocytes were isolated using retrograde aortic perfusion with a nominally calcium-free, collagenase buffer. In vitro ischemic preconditioning was induced by a 10-min episode of ischemic pelleting, followed by a 15-min post-incubation and a prolonged period of ischemic pelleting. Injury was assessed by determination of cell contracture and trypan blue permeability following hypotonic swelling and correlated with metabolic assays of lactate and adenine nucleotides. The protein phosphatase PP1/2A inhibitor calyculin A and PP2A-selective fostriecin protected isolated rabbit cardiomyocytes from lethal injury after a 10-min pre-incubation and when added late into ischemic pellets after a delay of 75 min. At the time of late drug addition, cells were severely ATP-depleted and in rigor contracture. Protection with Calyculin A from 1 nM to 1 microM was dose-related. Cells pre-incubated with 10 nM to 10 microM fostriecin 10 min prior to ischemic pelleting were protected with an EC50 approximating 71 nM, implying protection at a PP2A-selective dose. The selective protein kinase C inhibitor, calphostin C, blocked ischemic preconditioning protection but not protection from 1 microM calyculin A. Protection of severely ischemic cardiomyocytes following protein phosphatase inhibition appears not to require PKC activity or ATP conservation. Pre-incubation of cells with calyculin A induced high levels of phosphorylation in p38 mitogen activated protein kinase (MAPK), as compared to the ischemia-induced phosphorylation observed in the untreated group only at 30 min of ischemia, providing evidence of protein phosphatase activity in cardiomyocytes. Pharmacological protection in late ischemia has been demonstrated, but the mechanism of protection is undetermined.

Calcium tolerant pig and rabbit cardiomyocytes were isolated using retrograde aortic perfusion of nominally calcium-free collagenase. Preconditioning protocols used 1 or 3×10-min episodes of ischemic pelleting or pre-incubation with 100 μ m adenosine, followed by a 15-min post-incubation and 180–240-min ischemic pelleting. Control cells were incubated and washed in parallel with the experimental groups. Injury was assessed by determination of cell morphology, trypan blue permeability following osmotic swelling, lactate and HPLC analysis of adenine nucleotides. Preconditioned pig cardiomyocytes had a reduced rate of ischemic contracture, but protection occurred without conservation of ATP. Preconditioned rabbit cardiomyocytes were protected without significant changes in rates of ischemic contracture or ATP depletion. Incubation of ischemic cells with the protein phosphatase inhibitor, fostriecin, at PP2A-selective concentrations (0.1–10 μ m ), mimicked preconditioning in both rabbit and pig cardiomyocytes. In rabbits, the K ATP channel blocker, 5-hydroxydecanoate (5-HD), did not block preconditioning or fostriecin protection. In the pig, 5-HD blocked both preconditioning and fostriecin protection, with return of the rates of ischemic contracture to control. However, 5-HD was an effective blocker of protection only in early ischemia. Fostriecin mimicked preconditioning in the rabbit and the early responses of the preconditioned pig. Preconditioning appears associated with protein phosphorylation in both the rabbit and the pig, but major pathways leading to protection may differ in the two species.

Orlistat is a gastrointestinal lipase inhibitor used for weight reduction in obese individuals. Enteric hyperoxaluria caused by orlistat leads to oxalate absorption. Acute oxalate nephropathy is a rare complication of treatment with orlistat. Herein we report a patient presenting with acute renal failure which improved minimal with intravenous hydration. She was found to have oxalate crystals on renal biopsy. Patient admitted orlistat use over the counter for weight reduction on further questioning. The purpose of this case review is to increase awareness among patients since they are more focused on losing weight. This case also calls for the provider attention to educate patients regarding side effects of orlistat because of easy availability of orlistat over the counter.

Although adenosine receptors have been implicated in the induction of preconditioning in a variety of experimental models, there is controversy concerning the specific adenosine receptor subtypes mediating this effect. Concentration-protection relationships for adenosine and adenosine agonists in rabbit cardiomyocytes were used to characterize the role of adenosine receptor subtypes in preconditioning. Isolated cells were ischemically preconditioned or pre-incubated for 10 min with increasing concentrations of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), APNEA (N6-2-(4-aminophenyl)ethyladenosine), or BNECA (N6-benzyl-5'-N-ethyl-carboxamidoadenosine) in the presence or absence of 1 or 10 microM of the selective A1-adenosine antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine). Following a 30-min post-incubation period, cells were pelleted, layered with oil and ischemically incubated for 180 min. Injury was assessed by osmotic swelling and trypan blue exclusion of sequential samples, and determination of the areas beneath the mortality curves. Adenosine produced a broad concentration-protection curve which was displaced to the right by DPCPX. The curve for A1-selective agonist CCPA was biphasic, with an initial response below 1 nM and a second above 1 microM. DPCPX abolished the early response leaving a steep monophasic curve between 0.1 and 10 microM CCPA. The APNEA curve appeared moriophasic, the major slope occurring between 1-100 nM; DPCPX (1 microM) shifted the concentration-response curve approximately 30-fold and decreased the slope. Adenosine receptor agonist BNECA produced preconditioning characterized by a shallow monophasic concentration-protection curve with a maximal effect of 49% and an EC50 of approximately 5 nM; DPCPX shifted the BNECA concentration-protection relationship approximately 40-fold with only a modest increase in slope. Analysis of the data suggests that induction of preconditioning results from interaction of agonists with the A1 receptor and a second adenosine receptor having properties consistent with the A3 receptor. Adenosine, CCPA, APNEA, BNECA and DPCPX each appear to be selective for the A1 adenosine receptor subtype in isolated rabbit cardiomyocytes.

Objective: The aim was to discriminate among several hypotheses of preconditioning of isolated rabbit cardiomyocytes and to determine if ischaemic preincubation would evoke a protective response. Methods: Isolated myocytes were subjected to 5 min of preincubation, in the presence or absence of glucose, and incubated in the presence of 1 mM iodoacetic acid during the final sustained ischaemic period. In a second series, the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA), ingenol 3, 20-dibenzoate, and thymeleatoxin were added during preincubation. In a third series, preincubation periods were substituted by brief ischaemic pelleting of cells. Final prolonged ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury was determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 5 min glucose-free preincubation. Addition of iodoacetic acid into the final ischaemic pellet increased the rates of rigor contracture and injury, but did not abolish the protective response. Direct protein kinase C activation with PMA, a non-selective phorbol ester, and ingenol, an e, δ-PKC isozyme selective activator, protected cells, but thymeleatoxin, an α,β,γ-PKC isozyme selective activator, did not. A 10 min ischaemic preincubation preconditioned, but the protection was not enhanced when ischaemia was extended to 30 min, or when PMA was included during the initial ischaemic preincubation. Adenosine partially inhibited the response. Conclusions: (1) Preconditioning of isolated myocytes is not dependent on glycolysis or glucose transport. (2) Preconditioning appears dependent on activation of the e-PKC isoformn. (3) Ischaemia is capable of preconditioning isolated myocytes in vitro, and initiation of this effect is modified by simultaneous additional of adenosine but not by direct protein kinase C activation with PMA. Induction of protection by PMA and ingenol shows that protection requires protein kinase C activation, but direct potassium channel activation by regulatory G proteins is not critical. Cardiovascular Research 1994; 28 :1700-1706

Objective: The aim was to further characterise an experimental model of preconditioning of isolated rabbit cardiomyocytes and to determine the role of adenosine receptor subtypes in initiation of the protective response. Methods: Isolated myocytes were subjected to 5 min preincubation in the presence or absence of glucose and various agonists and antagonists of adenosine receptors. Ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury during ischaemia was determined by sequential sampling of the pelleted cells and assessment of trypan blue permeability following 85 mOsm swelling. Results: Myocytes were preconditioned with a 30-50% reduction of injury by a 5 min glucose-free preincubation. Substitution of 5 mM pyruvate for glucose during preincubation did not prevent the protective response. Protection was maintained over a 60-180 min postincubation period. Protection was blocked by 100 μM of the non-specific adenosine A1A2, antagonist SPT, both when added only during preincubation or only into the ischaemic pellet. Calphostin C, a specific protein kinase C inhibitor at 200 nM, added to the ischaemic pellet blocked protection. Preincubation with R-PIA, the adenosine A1 agonist, did not precondition at an A1 selective dose of 1 μM, but did at 100 μM. The selective A2 agonist CGS 12680 (1 μM) did not precondition. The selective A1/A3 adenosine agonist, APNEA, preconditioned at 1 μM and 200 nM dose levels. Preconditioning induced either by 200 nM APNEA or by glucose-free preincubation was not blocked by 200 nM or 10 μM of the A1 antagonist DPCPX, which has extremely low affinity for A3 receptors, but was blocked by 1 μM of the A1/A3 adenosine antagonist BW 1433U83. Conclusions: Preconditioning can be induced in isolated myocytes by a 5 min preincubation/30 min postincubation protocol, and a similar protection induced by adenosine agonists with A3, but not A1 selectivity. Preconditioning is blocked by non-selective or selective A1/A3 adenosine antagonists and a specific protein kinase C inhibitor, but not by A1 antagonists with little affinity for A3 receptors. The results suggest that preconditioning in isolated rabbit myocytes requires participation of adenosine receptors with agonist/antagonist binding characteristics of the A3 subtype, and is likely to be mediated by activation of protein kinase C. Cardiovascular Research 1994; 28: 1049-1056

Objective: The aim was to determine if isolated rabbit cardiomyocytes could be preconditioned. Methods: Cardiomyocytes isolated from rabbit hearts were subjected to 15 min oxygenated preincubation, with and without substrate, prior to concentration into an ischaemic slurry, with or without glucose present. The effects of an adenosine agonist (CCPA), an adenosine receptor blocker (SPT), and the protein kinase C blocker, calphostin C, on rates of ischaemic contracture and survival of the myocytes were determined after various times of ischaemia, following resuspension of the cells in hypotonic media. Results: A glucose-free preincubation period protected myocytes from subsequent ischaemic injury, with a 40% reduction of cell death at 90-120 min and 1-2 h delay in cell death. CCPA added during preincubation and during the ischaemic period also tended to protect from injury, but the differences were not significant and protection was less than with a glucose-free preincubation. Although preincubation with CCPA did not precondition, SPT added to the preincubation medium only, or to both the preincubation medium and the ischaemic pellet, inhibited the preconditioning effect of a glucose-free preincubation period. Calphostin C, added only into the ischaemic pellet, inhibited the preconditioning effect of glucose-free preincubation. Conclusions: Glucose-free preincubation protects ischaemic isolated myocytes from subsequent ischaemia. The degree of protection is great enough to account for protection seen in intact hearts, following preconditioning protocols. Protection is blocked by SPT and a highly specific protein kinase C inhibitor, calphostin C. Protection from ischaemic injury that seems to mimic ischaemic preconditioning can be induced in isolated cardiomyocytes, and appears dependent on adenosine receptors and activation of protein kinase C. Cardiovascular Research 1994; 28 :72-77

Objective: The aim was to determine if isolated rat cardiomycytes could be protected from ischaemic cell death by preincubation with adenosine or adenosine agonists. Methods: Cardiomyocytes isolated from rat hearts were preincubated in the presence of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), or carbachol prior to concentration into an ischaemic slurry. Effects of glycolysis and of isoprenaline were determined by addition of iodoacetic acid or isoprenaline to the ischaemic incubates and by exclusion of glucose from all media. Rates of ischaemic contracture were determined and survival of the myocytes versus paired control preparations was determined after various times of ischaemia, following resuspension of the cells in isotonic or hypotonic media. Results: Adenosine and CCPA produced only a small reduction of the rates of contracture and death of isolated myocytes. Carbachol gave no significant protection. Neither the degree of injury of control cells nor the amount of protection by CCPA was altered in the presence of added isoprenaline. Protection was abolished by the A1 receptor blocker sulphophenyl theophylline, iodoacetic acid, and exclusion of glucose. Conclusions: Adenosine and adenosine agonists afford a minimal degree of protection to ischaemic isolated myocytes by a glucose dependent mechanism. This protection does not appear to account for the larger degree of protection seen in intact hearts, following similar preconditioning protocols. The failure of adenosine to protect may be related to the quiescent state of isolated cardiomyocytes, or be species specific in that adenosine may not be the trigger for preconditioning in rats. Cardiovascular Research 1993; 27 :1670-1676

Isolated adult rat myocytes were subjected to 180 min of metabolic inhibition or incubated in ischaemic pellets, in the presence and absence of 10 μ m okadaic acid (OA) or calyculin A (CL-A). Contracture and viability was determined by light microscopic analysis of trypan blue-stained preparations and ATP levels by HPLC. Osmotic fragility was assessed by brief hypotonic swelling of cells in 170 or 85 mOsm media prior to determination of viability. Neither drug significantly affected the relatively rapid rates of contracture of myocytes during metabolic inhibition, and both afforded significant protection from development of trypan blue permeability and osmotic fragility. Both OA and CL-A significantly accelerated the rates of contracture and ATP depletion of myocytes during ischaemic incubations. Despite an enhanced rate of ATP depletion, which would be expected to accelerate development of injury, neither drug accelerated development of loss of viability or development of osmotic fragility as measured by 170 mOsm swelling. Mathematical compensation for different rates of ATP depletion confirmed that a protective effect of the drugs, during ischaemic incubation, was masked by their enhancement of the rate of injury, following swelling at 170 mOsm. When the effects of CL-A on ischaemic cells were examined at 85 mOsm, a more stringent test for osmotic fragility, protection was found without compensation for differing rates of ATP depletion. A dose/response curve for CL-A showed some effect at 100 n m and a nearly full effect during metabolic inhibition at 1 μ m concentrations. It is concluded that protein phosphatase inhibitors reduce the rates of development of osmotic fragility of metabolically inhibited cells and reduces the rate of injury relative to the rate of ATP depletion of ischaemic cardiomyocytes. Phosphorylation mechanisms may be important to development of irreversible myocardial cell injury.

Fatal abdominal sarcomatosis is a rare complication secondary to gastrointestinal stromal tumor (GIST) arising from the small intestine. Here, we describe a 49-year-old man who presented with massive ascites and multiple mesenteric solid masses. Autopsy showed large necrotic mass (19 x 14 x 8.0 cm) surrounding the terminal ileum with multiple smaller nodules on the omentum and mesentery. Histological examination revealed highly cellular, predominantly bland, spindle cells with low mitotic activity. Gastrointestinal stromal tumor was confirmed by strong positive staining for c-KIT (CD 117). Our case is an example of fatal intra-abdominal sarcomatosis of GIST where the histological features were essentially bland.

A loss of sarcolemmal dystrophin was observed by immuno-fluorescence studies in rabbit hearts subjected to in situ myocardial ischemia and by immuno-blotting of the Triton soluble membrane fraction of isolated rabbit cardiomyocytes subjected to in vitro ischemia. This ischemic loss of dystrophin was a specific event in that no ischemic loss of sarcolemmal alpha-sarcoglycan, gamma-sarcoglycan, alphaDG, or betaDG was observed. The maintenance of sarcolemmal betaDG (43 Kd) during ischemia was interesting in that dystrophin binds to the C-terminus of betaDG. However, during late in vitro ischemia, a 30 Kd band was observed that was immuno-reactive for betaDG. Additionally, this 30 Kd-betaDG band was observed in rabbit myocardium subjected to autolysis. Finally, the 30 Kd-betaDG was observed in the purified sarcolemmal fraction of rabbit cardiomyocytes subjected to a prolonged period of in vitro ischemia, confirming the sarcolemmal localization of this band. The potential patho-physiologic significance of this band was indicated by the appearance of this band at 120-180 min of in vitro ischemia, directly correlating with the onset of irreversible injury, as manifested by osmotic fragility. Additionally the appearance of this band was significantly reduced by the endogenous cardioprotective mechanism, in vitro ischemic preconditioning, which delays the onset of osmotic fragility. In addition to dystrophin, betaDG binds caveolin-3 and Grb-2 at its C-terminus. The presence of Grb-2 and caveolin-3 in the membrane fractions of oxygenated and ischemic cardiomyocytes was determined by Western blotting. An increase in the level of membrane Grb-2 and caveolin-3 was observed following ischemic preconditioning as compared to control cells. The formation of this 30 Kd-betaDG degradation product is potentially related to the transition from the reversible to the irreversible phase of myocardial ischemic cell injury and a decrease in 30 Kd-betaDG might mediate the cardioprotection provided by ischemic preconditioning.

Ischemic preconditioning (IPC) is an endogenous mode of cardioprotection that is induced by a cycle of brief ischemia and reperfusion. Ischemic preconditioning reduces infarct size in intact animal models, reduces reoxygenation necrosis and enzyme release in perfused hearts, delays the onset of osmotic fragility in isolated cardiomyocyte models and attenuates necrosis and apoptosis in cultured cardiomyocyte models. A variety of mechanisms have been proposed to mediate the cardioprotection provided by ischemic preconditioning. These include: 1) Activation of PKC and MAPK pathways; 2) Reductions in ischemic ATP depletion, electrolyte changes, or free radical generation; 3) Production of nitric oxide; and 4) Opening of the mitochondrial KATP channel. However, there is currently no clear consensus on the end-effector mechanism(s) responsible for the cardioprotection provided by ischemic preconditioning. A recent report indicates that ischemic preconditioning enhances cell volume regulation and reduces the swelling of isolated cardiomyocytes during simulated ischemia (24). Evidence relevant to the novel hypothesis that reduced cellular swelling may be an end-effector of preconditioning protection is briefly reviewed below.

A loss of sarcolemmal dystrophin was observed by immuno-fluorescence studies in rabbit hearts subjected to in situ myocardial ischemia and by immuno-blotting of the Triton soluble membrane fraction of isolated rabbit cardiomyocytes subjected to in vitro ischemia. This ischemic loss of dystrophin was a specific event in that no ischemic loss of sarcolemmal a-sarcoglycan, γ-sarcoglycan, αDG, or βDG was observed. The maintenance of sarcolemmal βDG (43 Kd) during ischemia was interesting in that dystrophin binds to the C-terminus of βDG. However, during late in vitro ischemia, a 30 Kd band was observed that was immun-reactive for βDG. Additionally, this 30 Kd-βDG band was observed in rabbit myocardium subjected to autolysis. Finally, the 30 Kd-βDG was observed in the purified sarcolemmal fraction of rabbit cardiomyocytes subjected to a prolonged period of in vitro ischemia, confirming the sarcolemmal localization of this band. The potential patho-physiologic significance of this band was indicated by the appearance of this band at 120–180 min of in vitro ischemia, directly correlating with the onset of irreversible injury, as manifested by osmotic fragility. Additionally the appearance of this band was significantly reduced by the endogenous cardioprotective mechanism, in vitro ischemic preconditioning, which delays the onset of osmotic fragility. In addition to dystrophin, βDG binds caveolin-3 and Grb-2 at its C-terminus. The presence of Grb-2 and caveolin-3 in the membrane fractions of oxygenated and ischemic cardiomyocytes was determined by Western blotting. An increase in the level of membrane Grb-2 and caveolin-3 was observed following ischemic preconditioning as compared to control cells. The formation of this 30 Kd-βDG degradation product is potentially related to the transition from the reversible to the irreversible phase of myocardial ischemic cell injury and a decrease in 30 Kd-βDG might mediate the cardioprotection provided by ischemic preconditioning. (Mol Cell Biochem 242: 71-79, 2003)

Sarcolemmal blebbing and rupture are prominent features of irreversible ischemic myocardial injury. Dystrophin and spectrin are sarcolemmal structural proteins. Dystrophin links the transmembrane dystroglycan complex and extracellular laminin receptors to intracellular F-actin. Spectrin forms the backbone of the membrane skeleton conferring an elastic modulus to the sarcolemmal membrane. An ischemic loss of membrane dystrophin and spectrin, in ischemically pelleted rabbit cardiomyocytes or in vivo 30--45 min permanently ischemic, LAD-ligated hearts, was detected by immunofluorescence with monoclonal antibodies. Western blots of light and heavy microsomal vesicles and Triton-extracted membrane fractions from ischemic myocytes demonstrated a rapid loss of dystrophin coincident with sub-sarcolemmal bleb formation, subsequent to a hypotonic challenge. The loss of spectrin from purified sarcolemma of autolysed rabbit heart, and both isolated membrane vesicles and Triton solubilized membrane fractions of ischemic cardiomyocytes correlated linearly with the onset of osmotic fragility as assessed by membrane rupture, subsequent to a hypotonic challenge. In contrast to the ischemic loss of dystrophin and spectrin from the membrane, the dystrophin-associated proteins, alpha-sarcoglycan and beta-dystroglycan and the integral membrane protein, sodium-calcium exchanger, were maintained in the membrane fraction of ischemic cells as compared to oxygenated cells. Preconditioning protected cells, but did not significantly alter ischemic dystrophin or spectrin translocation. This previously unrecognized loss of sarcolemmal dystrophin and spectrin may be the molecular basis for sub-sarcolemmal bleb formation and membrane fragility during the transition from reversible to irreversible ischemic myocardial injury.

The hypothesis that irreversible ischemic injury is related to sub-sarcolemmal blebbing and an inherent osmotic fragility of the blebs was tested by subjecting isolated control and ischemically preconditioned (IPC) or calyculin A (CalA)-pretreated (protected) rabbit cardiomyocytes to ischemic pelleting followed by resuspension in 340, 170 or 85 mosmol medium containing trypan blue. At time points from 0-240 min, osmotic fragility was assessed by the percentage of trypan blue permeable cells. Membrane blebs were visualized with India ink preparations. Bleb formation, following acute hypo-osmotic swelling, developed by 75 min and increased with longer periods of ischemia. Osmotic fragility developed only after 75 min. Cells resuspended in 340 mosmol media did not form blebs and largely retained the ability to exclude trypan blue, even after 240 min ischemia. Although the latent tendency for osmotic blebbing preceded the development of osmotic fragility, most osmotically fragile cells became permeable without evident sarcolemmal bleb formation. The onset of osmotic fragility was delayed in protected cells, but protection did not reduce the bleb formation. It is concluded that blebbing and osmotic fragility are independent manifestations of ischemic injury. The principal locus of irreversible ischemic injury and the protection provided by IPC may lie within the sarcolemma rather than at sarcolemmal attachments to underlying adherens junctions.

Alpha B Crystallin (alpha BC) is a putative effector protein of ischemic preconditioning (IPC), that is phosphorylated on Ser 45 by ERK1/2 and Ser 59 by the p38 MAPK substrate, MAPKAPK-2. Translocation and phosphorylation of alpha BC was determined in cytosolic and cytoskeletal fractions by 1D SDS-PAGE and IEF, or using Ser 45 and Ser 59 phospho-specific antibodies in: (1) control rabbit cardiomyocytes; (2) cells preconditioned by 10 min in vitro ischemia; or after pre-treatment with specific inhibitors of (3) Ser/Thr protein phosphatase 1/2A (calyculin A); (4) p38 MAPK (SB203580); or (5) ERK 1/2 (PD98059); all prior to 180 min ischemia. Ischemia induced a cytosolic to cytoskeletal translocation of alpha BC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of alpha BC were present in cytosolic but not cytoskeletal fractions at 0 min ischemia. By 60-90 min ischemia, D1/2 isoforms had translocated to the cytoskeletal fraction. Calyculin A maintained D1/2 levels throughout prolonged ischemia. SB203580 decreased alpha BC phosphorylation. Neither PD98059 nor IPC altered alpha BC phosphorylation during prolonged ischemia. It is concluded that alpha BC phosphorylation during ischemia is regulated by p38 MAPK but not by ERK 1/2. The inability to detect a correlation between IPC protection and either alpha BC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of alpha BC quantitated in this study are not protective end effectors of classical IPC.

Background —The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. Methods and Results —Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P P P Conclusions —Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 μ m adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60–180 min ischemia. Repetitive sub-maximal stimulations with 1 μ m adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 n m ) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 μ m ) or staurosporine (100 n m ) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.

This study was designed to test the hypothesis that induction of the preconditioned state results in a sustained translocation of protein kinase C (PKC) which accounts for the memory associated with preconditioning. Isolated rabbit cardiomyocytes were subjected to established preconditioning protocols using either adenosine or transient ischemia. At timed intervals during induction of preconditioning (PC), post-incubation or final sustained ischemia, cells were harvested, subjected to digitonin lysis and separated into cytosolic and particulate fractions. Samples were evaluated by Western blot analysis with monoclonal antibodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were quantified by densitometry. Internal controls for each experiment included oxygenated cardiomyocytes and cells with PKC translocation evoked by treatment with phorbol 12-myristate 13-acetate (PMA). For control oxygenated cells, the particulate fraction contained about 30% of PKC epsilon, 5–10% of PKC alpha and 60–70% of PKC zeta. Preconditioning with adenosine (100μm) or 10 min ischemia had no significant effect on these percentages. Furthermore, the relative amounts of the PKC isozymes associated with the particulate fraction of control and preconditioned cells did not differ after a post-incubation in oxygenated buffer or during a final ischemic incubation. PMA and ingenol completely translocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsilon. The distribution of PKC zeta between fractions was not affected by any drug. The protein phosphatase inhibitor calyculin A protected cells mimicking preconditioning. This protection was blocked by preincubation with the selective PKC inhibitor calphostin C but was largely retained if calphostin C was added only during the final ischemic period. It is concluded that PKC activity is required for preconditioning, but a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytesin vitro.

An intact heart is an integrated complex of tissues including the cardiomyocyte and interstitial elements (e.g. the extracellular collagen matrix, fibroblasts, mast cells, lymphatics, blood vascular elements and nerves). The complexity of the intact heart often excludes the reliable experimental isolation of single variables which influence cardiomyocyte physiology. The need for a simpler model led to development of methods to isolate cardiomyocytes. Initial attempts produced viable cells from neonatal and fetal hearts, but adult preparations were intolerant of calcium. Powell and Twist (1) first isolated of pure preparations of calcium tolerant adult myocytes. Cardiomyocytes have morphologic, metabolic, electrical and mechanical properties similar to those established for myocytes in intact hearts (2). In addition to the study of normal physiological processes, isolated cardiomyocytes have contributed significantly to understanding of the oxygen paradox, ischemia and reperfusion injury and preconditioning. An understanding of how in vitro cardiomyocyte models contribute to our understanding of cardiac disease might be facilitated by a brief review of these entities.

The cardiomyocyte cytoskeleton is composed of a highly organised complex array of specific proteins, arranged to transmit mechanical forces within the cell, to adjacent cells and the extracellular matrix, as well as to maintain internal organisation of cellular organelles. Although most of the published reports on cytoskeletal proteins refer to non-myocyte and smooth muscle cells, there seem significant homologies with cardiac structures. The specific interactions of some proteins in certain cytoskeletal structures are established and may be analogous to interactions in cardiac myocytes, but the roles of many proteins are uncertain, and the list of proteins that compose the cytoskeleton is likely to be incomplete. Some proteins may serve a dual role, contributing to signal transduction as well as to organisation and mechanical stability of the cell. Phosphorylation of cytoskeletal proteins, and elaborate cellular systems to control protein phosphorylation levels, suggest phosphorylation as a potential mechanism of controlling cytoskeletal assembly and remodelling. Disturbances of the cytoskeleton during ischaemia may produce alterations in cell structural integrity that could account for cell injury and death. Although mechanisms both of cytoskeletal assembly in normal cells and of cytoskeletal injury in ischaemic cells are currently poorly understood, research into the interactions of cytoskeletal proteins during ischaemia includes new approaches that may increase our understanding of the pathophysiology of the cardiac myocyte.

Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.

The relationship between myocardial cell contracture and injury during total metabolic inhibition (amylobarbital and iodoacetic acid) and ischemia was examined, using 5-50 mM butanedione monoxime (BDM) as an inhibitor of contracture. BDM had no apparent effect on control myocytes during 180 min incubations, but inhibited contracture following anoxia or ischemia in a dose-dependent fashion, as directly quantitated by length/width ratios. Cellular ATP levels decreased at a similar rate in the absence or presence of BDM, following metabolic inhibition. BDM-mediated inhibition of contracture was associated with accelerated cell injury, as defined by: the uptake of an extracellular marker (trypan blue) by the cardiomyocytes, by direct analysis of myoglobin released into the supernatant and by ultrastructural demonstration of defects in sarcolemmal membrane integrity. Calcium was not required for BDM's enhancement of injury, in that cells incubated in calcium free-EGTA buffer showed a similar BDM-mediated acceleration of injury. In the presence or absence of calcium, enhancement of injury was more marked in cells osmotically stressed with a brief incubation in hypotonic buffer, than in cells resuspended in isotonic media. It is concluded that BDM enhances development of osmotic fragility of inhibited or ischemic cardiomyocytes and that contracture is not a necessary contributing factor to myocardial cell death.

Isolated adult rat myocytes were used to develop an in vitro model of myocardial ischemia. Freshly isolated myocytes were spun into a cell pellet to limit extracellular volume. Excess supernatant was removed and the pellet was covered with mineral oil and incubated in a temperature controlled water bath. After various periods of incubation, cells were analyzed for adenine nucleotide levels, lactate accumulation, rate of cell death, and cell morphology. Adenine nucleotide profiles after 60 min incubation at 37 degrees C showed marked depletion of adenosine triphosphate (ATP) and large increases in adenosine monophosphate (AMP), adenosine, inosine, and lactate and no significant difference in levels of inosine monophosphate. These results are consistent with ischemic conditions. Reduction of the incubation temperature to 34 and 30 degrees C slowed the rate of cell squaring and the onset of cell death. Resuspension of ischemic cells after 30, 45, 60 and 90 min incubation in hypotonic buffer (170 mosmol) to induce acute cell swelling caused an increase in the number of non-viable cells at each time point. Control cells and ischemic cells incubated less than 30 min did not show increases in non-viable cells when subjected to hypotonic swelling. Morphological analysis revealed that isolated myocytes respond to ischemia in a heterogeneous fashion and exhibit changes at both light and electron microscopic levels similar to those seen in other ischemic models. These results indicate that pelleted isolated adult rat myocytes may be a useful in vitro model to study myocardial ischemic cells injury.

Acute myocardial cell death is associated with complex biochemical and morphological changes which depend upon the type of cell injury sustained by the myocardium. Of the many changes associated with acute ischemic or anoxic myocardial cell death, intracellular calcium overload is a prominent feature of ischemia-reperfusion injury as has been shown in many experimental models. Consequently, early hypotheses and investigative research regarding acute myocardial death centered around calcium overload and determining the events and/or series of events which lead to cellular calcium overload. During our investigations of the mechanism(s) of cell damage associated with cellular injury, evidence appeared that cellular contracture and/or physical stress may be involved with acute cellular death in some experimental systems. Recently there is a growing body of evidence to suggest that cells irreversibly injured by hypoxia or ischemia may become fragile and therefore not able to withstand physical stresses associated with reperfusion or reoxygenation. This suggests that calcium overload may not be the primary cause of cell death. This chapter will evolve a discussion of the role of mechanical factors in the development of irreversible myocardial cellular injury and cell death.

The role of microvascular damage in the genesis of the "no-reflow" phenomenon was investigated in the left ventricular myocardium of dogs subjected to temporary occlusions of a major coronary artery for 40 and 90 min. Intravenous carbon black or thioflavin S (a fluorescent vital stain for endothelium) were used to demonstrate the distribution of coronary arterial flow in control and damaged myocardium. These tracers were injected simultaneously with release of the coronary occlusion or after 5 or 20 min of reflow of coronary arterial blood. After 40 min of ischemia plus arterial reperfusion, usually the tracers were evenly distributed throughout the damaged tissue at each time of reperfusion. On the other hand, when reflow was allowed after 90 min of ischemia, portions of the inner half of damaged myocardium were not penetrated by the tracers. Electron microscopic study of this poorly perfused tissue revealed severe capillary damage; endothelial cells with large intraluminal protrusions and decreased pinocytic vesicles were common. Also, occasional intraluminal fibrin thrombi were noted, as well as extravascular fibrin deposits and erythrocytes. Myocardial cells were swollen in both poorly perfused and well-perfused irreversibly injured tissue. Contraction bands and mitochondrial Ca(2+) accumulation were prominent features of irreversible injury with reflow at 40 min but were not noted after 90 min of ischemia in areas with poor perfusion. These results suggest that 40 min of ischemia were tolerated by the capillary bed of the dog heart without serious capillary damage or perfusion defects, but that 90 min of ischemic injury was associated with the "no-reflow" phenomenon, i.e., failure to achieve uniform reperfusion. This failure of reflow was associated with extensive capillary damage and myocardial cell swelling. Death of severely ischemic myocardial cells in this model occurs before the onset of capillary damage and the no-reflow phenomenon.

Substrate-free anoxic perfusion of isolated rat hearts results in the release of creatine phosphokinase (CPK) into the coronary effluent. To test the specificity of this response a series of experiments was undertaken, using isolated rabbit and guinea-pig hearts instead of rat hearts. A marked species variation was found not only in the CPK release pattern but also in the protective effect of glucose, and the effect of reoxygenation.

d -Serine and d -2,3-diaminopropionic acid ( d -DAPA) induce necrosis of the epithelial cells of the proximal straight tubules of the rat kidney. Nineteen compounds with chemical structures and/or functional groups similar to d -serine or d -DAPA were tested for their protective effect against the induction of this injury. The acute tubular necrosis inducible by d -serine and d -DAPA was prevented by d -alanine, d -threonine, d -homoserine, dl -α-methyl serine, β-hydroxy- dl -leucine, and α-aminoisobutyric acid. In addition, protection from necrosis due to d -DAPA, but not that due to d -serine, was found with l -threonine, l -serine, l -2,3-diaminopropionic acid, and 3-hydroxypropionic acid. Little or no protective effect against d -serine of d -DAPA was afforded by l -alanine, β-alanine, dl -isoserine, dl -3-hydroxybutyric acid, l- or dl -lactic acid, 2-amino-2-methyl-1-propanol, taurine, and isethionic acid. The compounds that protected against d -serine-induced necrosis had in common the d -configuration (except for aminoisobutyric acid), the α-amino group, and the carboxyl group. The structural characteristics and chirality of the protective agents against d -DAPA were more varied and less definable than for d -serine. d -Alanine, dl -α-methyl serine, and α-aminoisobutyric acid protected on a mole-for-mole basis against d -serine-induced necrosis. d -Alanine was protective against d -serine even when given 30 min after d -serine administration. These results suggest that protection may be due to competition with d -serine or d -DAPA for a cellular or transport binding site.

The effects of physical stresses produced by transient distension of a left ventricular balloon, on myocardial creatine kinase (CK) release and cellular morphology, were studied in Langendorff perfused rat hearts at 37 degrees C. Hearts subjected to 15 or 30 min anoxia, developed anoxic contracture, but only small amounts of CK were released following ventricular distension. In contrast, when anoxic hearts in contracture were perfused with calcium-free medium for 5 min, prior to distension, there occurred a large peak of CK release immediately following inflation of the intraventricular balloon. Oxygenated hearts or hearts made anoxic, but which had not developed contracture, release little CK activity, although they were subjected to ventricular distension after a calcium-free perfusion period. Large myocardial enzyme releases were associated with morphological lesions of widely separated cells, dehiscence of intercalated disc cell junctions, and sarcolemmal membrane damage. Hearts subjected to calcium-free perfusion were abnormally susceptible to physical stress-induced myocardial enzyme release.

The effects of polyethylene glycol (PEG), mannitol and dextran on the osmotic properties of solutions, and the effects of hyperosmolar solutions of these oncotic agents on swelling of tissue slices and creatine phosphokinase (CPK) release from Langendorf-perfused rat hearts were studied under oxygenated and anoxic conditions and after reoxygenation following an anoxic interval. Mannitol solutions exhibited nearly ideal osmotic behavior, while both solutions of dextran and PEG showed marked deviations from ideal behavior. Hypertonic dextran and PEG solutions prevented both anoxic and cold-induced swelling of tissue, while mannitol only slightly reduced tissue swelling. Anoxic enzyme release was reduced by PEG, but not by dextran. Hypertonic perfusion media containing either mannitol or dextran, but not PEG, caused cell damage and CPK release from rat hearts during oxygenated perfusions. All these oncotic agents reduced both cell swelling and oxygen-induced CPK release, suggesting that cell swelling may be important in mediating this injury. The role of cell swelling in anoxic enzyme release cannot be determined from these experiments because of the possible superimposed cytotoxic effects of dextran and mannitol.

The effects of anoxia and calcium-free perfusion on enzyme release, were studied in perfused rat hearts, at 37°C. In oxygenated hearts, calcium-free medium caused detachment of cells at intercalated discs, but no cell separation or enzyme release. Sixty minutes of anoxia with calcium-free medium caused striking cell separations, contracture of sarcomeres and enzyme release. Anoxia, followed by calcium-free medium, produced a sudden, energy-independent enzyme release with contracture and cell separations. Contraction bands as well as cell separations, were present in anoxic hearts reoxygenated in the absence of calcium. It is proposed that calcium-free media causes loss of integrity of intercalated disc junctions and contraction, and anoxic contracture pulls cells apart at weakened cell junctions, mediating membrane damage and enzyme release.

The pathogenesis of drug-induced renal cystic disease. Oral administration of 2-amino-4,5-diphenyl thiazole HCl to rats over a period of 11 weeks produced marked, diffuse, progressive renal cystic disease without significant tubular obstruction or interstitial fibrosis. Light and electron microscopic and microdissection studies demonstrated that cystic changes began in collecting ducts in the medulla and extended to cortical collecting and distal tubules and loops of Henle. Micropuncture studies on pair-fed control animals treated with the drug for five weeks and on pair-fed controls revealed that glomerular function was unaltered and that intratubular pressure and reabsorption of solute and water along proximal convoluted and distal tubules were normal in cystic kidneys. These data indicate that nephrons with cystic change are capable of normal function. In the absence of tubular obstruction, it is proposed that cystic disease was due to a drug-induced structural defect of the tubular basement membrane which in the presence of a normal transtubular pressure gradient results in progressive tubular dilatation. Pathogenie de la maladie renale kystique induite par une drogue. L'administration orale de 2-amino-4,5-diphenyl thiazole HCl a des rats pendant une periode de 11 semaines produit une maladie kystique intense, diffuse et progressive sans obstruction tubulaire ou fibrose interstitielle significatives. La microscopie optique et electronique et la microdissection montrent que les modifications kystiques atteignent d'abord les canaux collecteurs dans la medullaire et s'etendent ensuite aux canaux collecteurs corticaux, aux tubes distaux et aux anses de Henle. L'etude par microponctions d'animaux traites pendant cinq semaines et de controles nourris de la meme facon montre que la fonction glomerulaire n'est pas alteree, que la pression intratubulaire et la reabsorption d'eau et de substances dissoutes le long des tubes contournes proximaux et des tubes distaux sont normales dans les reins kystiques. Les observations indiquent que les nephrons atteints de modifications kystiques sont susceptibles d'un fonction normale. En l'absence d'obstruction tubulaire il est propose que la maladie kystique soit due a un defaut structural induit par la drogue, de la membrane basale tubulaire qui, en presence d'un gradient de pression transtubulaire normal, a pour consequence une dilatation tubulaire progressive.

Beagle dogs, 24 ± 6 months old, fed a thiouracil-free semi-synthetic diet containing hydrogenated coconut oil and cholesterol (SS diet) for 12 months, developed marked hyperlipidemia and severe atherosclerosis. SS diet produced a marked elevation of serum cholesterol, triglyceride, phospholipid, and β-lipoprotein and severe atherosclerosis in large and small arteries. Intimal fatty lesions were always present in the abdominal aorta and many of its branches. Large and small coronary arteries showed similar lesions. The degree of atherosclerosis was directly related to circulating lipid levels. Dextrothyroxine, at dose levels of 0.1 (equivalent to normal human dose) and 0.5 mg/kg body weight, produced a significant dose related lowering of serum lipids and was associated with a markedly decreased severity of aortic and coronary artery lesions. Untreated control dogs that were maintained on purina dog meal developed nwither hyperlipidemia nor atherosclerosis.