Thermal and endocrine regulation of gonadal apoptosis during sex differentiation in pejerrey Odontesthes bonariensis.

The fate of the differentiating gonads in pejerrey Odontesthes bonariensis is determined by the environmental water temperature experienced by the larvae during the critical period of sex determination. We previously reported a link between apoptosis, temperature and sex differentiation in this species. To clarify this link, we subjected larvae to thermal and endocrine treatments between hatching and the onset of histological sex differentiation of the gonads and assessed the patterns of gonadal development and apoptosis by light microscopic histology and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, respectively. Apoptotic labeling was widespread among somatic cells of the anterior region of the right gonads of fish reared at the male-producing temperature (MPT) and part of the fish at the mixed sex-producing temperature prior to sex differentiation. In contrast, TUNEL-positive cells were rarely observed in gonads at the female-producing temperature (FPT). Administration of exogenous estrogen completely prevented MPT-induced masculinization, induced feminization and reduced gonadal apoptosis, whereas an aromatase inhibitor (fadrozole) induced TUNEL signals in the gonads of FPT-reared larvae. These results provide strong evidence that apoptosis in somatic cells in the right lobe of the gonads might play a key role in testicular differentiation in pejerrey and that estrogens are involved in the regulation of this process.

Renal impairment after laparoscopic radical nephrectomy affects hypoglycaemic therapy.

WHAT IS KNOWN AND OBJECTIVE: Renal impairment is unavoidable after laparoscopic radical nephrectomy (LRN) and is an important consideration for drug therapy. It is possible that the renal impairment after LRN causes adverse reactions following reduced elimination of some renally excreted drugs, such as hypoglycaemic drugs. However, there are few studies of renal function in patients with diabetes mellitus (DM) in the first week after LRN. The purpose of this study was to examine whether renal impairment after LRN affected glycaemic control. We assessed pre- and postoperative renal function of DM patients and examined whether re-administration of hypoglycaemic drugs in the first week after LRN causes episodes of hypoglycaemia. METHODS: Renal carcinoma patients undergoing LRN in Nagoya University Hospital from January 2007 to December 2009 were identified in a retrospective cohort study design. Patients were divided into non-DM (n = 60) and DM (n = 14) groups. RESULTS AND DISCUSSION: There were significant differences in postoperative estimated glomerular filtration rate values between the non-DM and DM groups. Four of nine patients (44%) experienced hypoglycaemia induced by re-administration of hypoglycaemic drugs, namely, sulfonylureas. WHAT IS NEW AND CONCLUSION: In the present study, we found the first evidence that renal impairment in the first week after LRN was a risk factor of hypoglycaemia. To prevent hypoglycaemia after LRN, assessment of renal function and the use of insulin therapy are important.

[Solitary fibrous tumor of the seminal vesicle]. / Solitärer fibröser Tumor der Samenblase.

A 56-year-old man presented to our hospital with a pelvic mass. The tumor was diagnosed to occur from right seminal vesicle and to be a benign solitary fibrous tumor by transrectal tumor biopsy. The tumor enlarged during follow up, and he under-went resection of the tumor.

Ewing's sarcoma / primitive neuroectodermal tumor of the kidney.

A 42-year-old female presented with right back pain. The CT scan revealed a 72-mm space-occupying lesion in the middle portion of the right kidney. No metastasis was proven. She underwent laparoscopic radical nephrectomy and lymph node disection. The histopathological examination revealed a high-grade primitive small round tumor the cells of which were strongly positive for CD99 and vimentin. Fluorescence in situ hybridization analysis using a DNA probe for the Ewing sarcoma breakpoint region 1 (EWSR 1) on chromosome 22g12 revealed a rearrangement of the EWSR 1 locus. The diagnosis was Ewing's sarcoma / primitive neuroectodermal tumor of the kidney. She underwent 13 cycles of chemotherapy, and has no evidence of recurrence 19 months after surgery.

Efficiency of laparoscopic-assisted renal biopsy.

BACKGROUND: This study was made to present our experience and results with transperitoneal laparoscopic-assisted renal biopsy (LARB) in Nagoya University Hospital as a good alternative for open renal biopsy. METHODS: 21 patients (14 male, 7 female, mean age 58 years, range 21-83 years) were unsuitable for percutaneous renal biopsy. Therefore, they underwent laparoscopic-assisted renal biopsy. The kidney was approached transperitoneally via three ports, cortical tissue was obtained using a 16-gauge gun-mounted semiautomatic biopsy needle. Hemostasis was obtained by applying pressure on the renal puncture using gauze until bleeding had been stopped (range 5-20 min). RESULTS: Adequate cortical tissue and accurate diagnoses were obtained in all patients. Mean operative time was 83 min (range 65-120 min) and mean estimated blood loss was 5.5 ml (range 1-10 ml). There were no intraoperative complications: no open conversion, blood transfusions or gross hematuria. All patients walked about freely and could tolerate regular food on the first postoperative day. The only postoperative complication was a hernia formation at the place of trocar insertion 3 months after the operation in one patient who previously underwent multiple surgery for 3 arterial grafts and appendicitis. CONCLUSIONS: LARB is a safe and accurate procedure for obtaining cortical biopsies with minimal blood loss. Although LARB remains a surgical procedure which requires general anesthesia, LARB to date may be considered as a good alternative to open renal biopsy for patients in whom a closed percutaneous approach is either a relative or absolute contraindication.

Conversion from mycophenolate mofetil to mizoribine for patients with positive polyomavirus type BK in urine.

It is known that administration of mycophenolate mofetile (MMF) is associated with BK virus (BKV) nephropathy in renal transplant recipients. To determine any inhibitory effect of mizoribine for BKV, seven patients with positive BKV in their urine who took MMF as immunosuppressive therapy were evaluated after MMF was changed to mizoribine. Baseline BKV DNA in urine, which ranged from 2.2 x 10(2) to 5.5 x 10(6) copies per milliliter, decreased in all cases (mean = 1.9 x 10(-1) times; median 2.8 x 10(-3) times). Four cases turned negative within 6 months and one within 12 months. No acute rejection or deterioration of graft function occurred during the administration of mizoribine. An inhibitory effect of mizoribine on BKV was suggested.

Dimorphic expression of dmrt1 and cyp19a1 (ovarian aromatase) during early gonadal development in pejerrey, Odontesthes bonariensis.

The pejerrey (Odontesthes bonariensis) is a teleost fish with strong temperature-dependent sex determination (TSD). Several studies have shown that dmrt1 and gonadal aromatase (cyp19a1) are implicated in the sex differentiation process in teleosts but little is known on the expression balance and endocrine regulation of these two genes during TSD. This study was designed to clarify the expression patterns of both genes during gonadal sex differentiation of pejerrey reared at female-, male- and mixed-sex-producing temperatures (FPT, MPT, and MixPT, respectively). The expression of dmrt1 was found to be significantly higher during gonadal sex differentiation at MPT compared to FPT. Conversely, cyp19a1 expression clearly increased during differentiation at FPT but not at MPT. The expression of both genes at MixPT showed a dimorphic profile with individual values resembling either those at the MPT or FPT. Administration of exogenous 17beta-estradiol down- and up-regulated the expression of dmrt1 and cyp19a1, respectively, regardless of temperature, and rescued the female phenotype at the MPT. However, treatment with the aromatase inhibitor Fadrozole caused masculinization without changing the pattern of gene expression. These results are strong evidence of the involvement of both genes in the gonadal differentiation process of pejerrey. The involvement of estradiol is discussed.

Autonomous catheter insertion system using magnetic motion capture sensor for endovascular surgery.

BACKGROUND: In order to reduce fluoroscope usage in endovascular surgery, there is a need to develop autonomous catheter insertion systems. METHODS: We propose a system for tracking the position and speed of a catheter using a magnetic motion capture sensor to provide feedback to a catheter-driving mechanism, to perform autonomous catheter insertion in major vasculature. Catheter insertion speed control and path reconstruction experiments were performed with the system inside a silicone model of major vasculature to simulate surgery. RESULTS: The system controlled the catheter for speeds of 6.14 mm/s and reproduced a two-dimensional path inside the silicone blood vessel phantom with less than 7 mm of error. CONCLUSIONS: We found that error in speed control rises as a result of friction between the catheter and the model wall. Path reconstruction error depends on the model's cross-sectional diameter, the properties of the catheter insertion mechanism, the magnetic sensor and the system guidance technique.

Laparoscopic radical nephrectomy for renal cell carcinoma.

PURPOSE: To estimate the efficacy of the laparoscopic radical nephrectomy we analyzed the clinical data of our series. PATIENTS AND METHODS: One hundred eighty five patients were enrolled in our laparoscopic radical nephrectomy program between July, 1992 and July, 2001. Of the 185 patients, 146 had small renal tumors (smaller than 5 cm in diameter) and 39 had large tumors (equal to or more than, 5 cm in diameter). Under a laparoscope the kidney, adrenal gland, and perirenal fatty tissue were dissected in an en bloc fashion. In case of taking out a small tumor, the specimen was fractionated within the sack to avoid an additional skin incision after entrapping in the laparoscopy sack in the working space. In case of a large tumor, regional lymph nodes dissection was done and the specimen was taken out intact in the sack through an enlarged incision. RESULTS: Our laparoscopic procedure was successful in 171 of the 185 cases; 14 patients required open surgery because of bleeding from an injured vessels or treatment for other injured organs. The mean operative time was 4.7 hours for both small and large tumors. Estimated blood loss was between 237 and 380 ml on average for small and large tumors, respectively. Full convalescence was achieved around 3 weeks after operation in both groups. Only one patient who had large tumor was found to have micrometastasis in 1 of 5 regional lymph nodes. Recurrences were observed in 4 cases of the small tumor group and in 2 cases of the large tumor group during 1 to 108 months of follow-up. CONCLUSION: Laparoscopic radical nephrectomy is a very useful and safe surgical procedure for renal cell carcinoma.

Role of STAT3 in ischemic preconditioning.

We recently demonstrated that ischemic preconditioning (IPC) induced by cyclic episodes of short durations of ischemia and reperfusion potentiates a signal transduction cascade involving protein tyrosine kinases and MAP kinases. A rapid activation of janus kinase (JAK) and several signal transducers and activators of the transcription (STATs) including STAT3, STAT5A and STAT6 has been shown to occur during myocardial ischemia and reperfusion. This study sought to examine if JAK/STAT signaling pathway play any role in classical early phase of IPC. Isolated working rat hearts were perfused for 15 min with KHB buffer in the absence or presence of a JAK kinase inhibitor tyrphostin AG490 (5 microm) followed by IPC, 30 min global ischemia and 2 h of reperfusion. The results demonstrated extensive phosphorylation of JAK2 and STAT3 in the IPC hearts which was almost completely abolished by an inhibitor of JAK2, AG490. IPC displayed cardioprotection as evidenced by improved post-ischemic contractile recovery, decreased myocardial infarct size and reduced number of apoptotic cardiomyocytes. AG490 blocked IPC-mediated cardioprotection by altering the IPC-mediated survival signal into death signal. Thus, IPC-induced upregulation of antiapoptotic gene bcl-2 and downregulation of pro-apoptotic gene bax are decreased and increased, respectively, in the AG490 treated hearts. The results suggest that early phase of IPC potentiates JAK/STAT signaling by activating STAT3 which transmits a survival signal to the myocardium.

[Laparoscopic radical prostatectomy--initial 10 cases].

PURPOSE: We report the clinical results and efficacy of laparoscopic radical prostatectomy for localized prostate cancer. PATIENTS AND METHODS: Between December, 1999 and June, 2000 we performed transperitoneal laparoscopic radial prostatectomy on 10 patients with T1 or T2 organ confined prostate cancer according to the techniques as described by Guillonneau et al. Different points were as follows: 1) We placed double J catheters during surgery to prevent ureteral injury. 2) We treated dorsal vein complex using an Endo-GIA-stapler. 3) We dissected the bladder neck from the prostate using an ultrasound scalpel in the manner to preserve the bladder neck. RESULT: We could not completed laparoscopic prostatectomy on two patients because of massive bleeding from dorsal vein and consuming too much time to suture urethra-bladder anastomosis. The average operating time in all cases was 8.1 hours including 1.8 hours in laparoscopic pelvic lymphadenectomy. The average estimated blood loss in all cases was 859 ml. There were 2 surgical complications with bladder injury and port site hernia. The histological examination revealed prostate cancer; pT2pNO in 8, pT3pNO in 2. The surgical margin and lymph nodes were negative in all patients. The duration of an indwelling catheter ranged from 5 to 40 days mean 17 days. The duration to recovery of normal micturition was 6 to 90 days, mean 40 days. CONCLUSION: Long-term follow-up and extensive studies are necessary to evaluate the efficacy of this procedure. It might provide shorter duration of an indwelling catheter and earlier recovery of normal micturition as compared with the conventional open surgery.

[Peripheral blood stem cell harvest for patients with germ cell tumors].

From January 1996 to December 1999, fifteen patients with germ cell tumors underwent peripheral blood stem cell harvest during 15 courses of bleomycin, etoposide, cisplatin (BEP), 4 courses of etoposide, ifosfamide, cisplatin (VIP) and 3 courses of high-dose etoposide mobilization at Nagoya University Hospital. We performed 29 aphereses during BEP, eight during VIP, and six during high-dose etoposide. Although we were able to harvest 4.4 x 10(6)/kg of median CD34 positive cells per apheresis during BEP, the number of stem cells (more than 4 x 10(6)/kg of CD34 positive cells), which are needed for tandem high-dose chemotherapy, could not be obtained during four courses of BEP. For three patients in whom white blood cell counts at nadir were 2,000/microL or more, however, the required number of CD34 positive cells were harvested. VIP provided only 1.7 x 10(6)/kg of median CD34 positive cells per apheresis, while, 7.3 x 10(6)/kg of CD34 positive cells were harvested during high-dose etoposide mobilization. The dose of G-CSF was a significant factor for the number of CD34 positive cells harvested during BEP (p = 0.02); however, there might be some relationship between the harvest and the number of the peripheral white blood cells on the day of apheresis (p = 0.08), the day to start G-CSF (p = 0.13), or the day to initiate apheresis (p = 0.27). Based on our experience, it is recommended that 5 micrograms/kg of G-CSF should be started from the 14th or 15th day of BEP until the last apheresis and that aphereses should be performed between the 19th and 21st day, especially at the days when the peripheral white blood cell count increases beyond 10,000/microL.

An essential role of the antioxidant gene Bcl-2 in myocardial adaptation to ischemia: an insight with antisense Bcl-2 therapy.

Reperfusion of ischemic myocardium results in apoptotic cell death, which can be blocked by adapting the heart to ischemic stress induced by cyclic episodes of brief periods of ischemia and reperfusion. In concert, the antiapoptotic gene bcl-2 is decreased by ischemia/reperfusion, but increased in the ischemically adapted myocardium. To examine if bcl-2 plays a crucial role in cardioprotection, adaptive cardioprotection was further examined in the hearts treated with antisense bcl-2 oligodeoxynucleotides (ODN). Isolated Langendorff-perfused rat hearts were divided into three groups: control (perfused with Krebs-Henseleit bicarbonate buffer for 210 min); 30-min ischemia followed by 2-h reperfusion; ischemic adaptation followed by 30-min ischemia and 2-h reperfusion. The last (adapted heart) group was subdivided into another two groups: one was transfected 48 h earlier with antisense bcl-2 ODN, whereas the other group was transfected with sense bcl-2 ODN. Cardioprotection was examined by determining cardiomyocyte death due to necrosis and apoptosis. Antisense gene therapy almost completely abolished bcl-2 protein expression in the hearts. Bcl-2 mRNA was down-regulated in the ischemic/reperfused heart, but up-regulated in the adapted myocardium. Adapted myocardium showed decreased infarct size and reduced number of apoptotic cardiomyocytes. Ischemia/reperfusion resulted in increased oxidative stress as evidenced by increased malonaldehyde formation. Adapted myocardium had a reduced amount of malonaldehyde. Antisense bcl-2 ODN completely abolished the cardioprotective effects of adaptation by eliminating the antideath signal of bcl-2. In concert, reduced oxidative stress in the adapted myocardium no longer persisted. The results suggest an antioxidant role of bcl-2 that appeared to be essential for the cardioprotection achieved by ischemic adaptation.

Experimental study of pulmonary artery infusion with cisplatin in a solitary pulmonary tumor model using a rat colorectal adenocarcinoma cell line.

OBJECTIVES: We assessed a tumor model prepared by open lung injection to study metastatic lung tumors, and evaluated the efficacy of pulmonary artery infusion. METHODS: Subjects were 30 male F344 rats. In experiment 1, we evaluated chemosensitivity of a rat colorectal adenocarcinoma cell line (RCN-9) using a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. In experiment 2, we injected RCN-9 cells into the left lung on day 0; on day 10, we measured tumor tissue blood flow before and after pulmonary arterial occlusion. In experiment 3, we injected RCN-9 cells into the left lung and conducted no further procedures in controls. The pulmonary artery infusion group underwent pulmonary artery infusion with 0.1 mg of cisplatin on day 3 and the sham group injection with saline solution alone. On day 10, rats were sacrificed and maximum tumor cross-section measured. RESULTS: In experiment 1, the drug concentration required to inhibit cell growth 50% was 2.45 x 10(-6) M. In experiment 2, tumor tissue blood flow decreased significantly after arterial occlusion (p = 0.003). In experiment 3, the maximum tumor cross-section in the pulmonary artery infusion group was significantly smaller than in shams (p = 0.0027) and controls (p = 0.0019). CONCLUSIONS: The pulmonary artery supplies tumors with blood, so this model appears useful in studying metastatic lung tumors, whose size was reduced significantly by pulmonary artery infusion with cisplatin. Pulmonary artery infusion is thus a promising modality in metastatic lung tumor treatment.

Insulin-like growth factor 1 prevents neuronal cell death and paraplegia in the rabbit model of spinal cord ischemia.

OBJECTIVE: Insulin-like growth factor 1 has been shown to be cytoprotective against ischemia-reperfusion injury in various organs. However, spinal cord protection by insulin-like growth factor 1 has not been tested. We have therefore examined the effect of insulin-like growth factor 1 on neuronal cell death and motor function after spinal cord ischemia. METHODS: Japanese white rabbits were subjected to spinal cord ischemia by clamping the abdominal aorta for 15 minutes. Insulin-like growth factor 1 (0.3 mg/kg) at a dose equipotent to insulin (0.3 IU/kg) in lowering blood glucose level or the control (phosphate-buffered saline solution as a vehicle) was administered intravenously 30 minutes before the aortic clamp. RESULTS: Hind-limb motor function had recovered normally 48 hours after the operation in all the rabbits (n = 8) treated with insulin-like growth factor 1. In contrast, all the control-treated (n = 8) and all but one of the insulin-treated (n = 6) rabbits had deteriorated to paraplegia by 48 hours after the operation. Histopathologic sections in the involved spinal cord segment showed that a significantly (P <.0001) greater number of motor neuron cells were preserved in the rabbits treated with insulin-like growth factor 1 (17.9 +/- 4.8 per section) than in those treated with the control (8.0 +/- 2.1). Although insulin was equipotent to insulin-like growth factor 1 in preserving the number of motor neuron cells (18.5 +/- 2.7), the percentage of motor neuron cells positive for terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling were significantly (P <.01) smaller in the rabbits treated with insulin-like growth factor 1 (6.0 +/- 4.6) compared with those treated with the control (54.6 +/- 33.8) and insulin (26.2 +/- 11.7). Immunohistochemical studies revealed that insulin-like growth factor 1 increased expression of the antiapoptotic Bcl-xL protein and inhibited expression of the proapoptotic Bax protein in motor neuron cells 24 and 48 hours after the operation. In contrast, expression of only Bax was increased after the operation in other groups of rabbits subjected to spinal cord ischemia. CONCLUSIONS: These results suggest that insulin-like growth factor 1, but not insulin with a conventional dose, protects motor neuron cells from ischemic spinal cord injury associated with differential regulation of Bcl-xL and Bax protein.

The long-term outcome of laparoscopic radical nephrectomy for small renal cell carcinoma.

PURPOSE: To evaluate the efficacy of laparoscopic radical nephrectomy in patients with small renal cell carcinoma, we analyzed the long-term results in those treated with laparoscopy and those undergoing open surgery. MATERIALS AND METHODS: A total of 149 patients with tumors less than 5 cm. in diameter enrolled in a radical nephrectomy program between January 1992 and March 2000. Of these patients 103 were treated laparoscopically and the remaining 46 underwent open surgery. Patient followup was until June 30, 2000. RESULTS: Laparoscopy followup was from 3 to 95 months (median 29). A total of 100 patients survived, 2 died without any recurrent disease in months 34 and 45, respectively, and 1 dropped out in postoperative month 3. Seeding of the port sites did not develop in any of the patients. There were 3 patients who had metastatic disease in months 3, 19 and 61, respectively, and 1 had local recurrence in postoperative month 43. The 5-year disease-free and patient survival rates were 95.1%, and 95.0%, respectively. Except for 2 patients who dropped out in months 10 and 16, respectively, 44 who underwent open surgery were followed from 11 to 101 months (median). Of the 44 patients 41 survived without any recurrent disease, 1 also survived with metastasis and 2 died of metastatic disease in months 7 and 11, respectively. The 5-year disease-free and patient survival rates were 89.7% and 95.6%, respectively. CONCLUSIONS: Laparoscopic radical nephrectomy can be an alternative to open nephrectomy in patients with localized small renal cell carcinoma.

Patch repair of postinfarction pseudo- and subepicardial aneurysm of the left ventricle.

Left ventricular pseudo-aneurysm and subepicardial aneurysm are rare complications of myocardial infarction. However, the prognosis of medical treatment is extremely poor and the operative procedure is controversial. We experienced 3 consecutive patients with this unusual left ventricular aneurysm, and described the clinical presentation, operative procedure and its result in these patients. The interval from myocardial infarction to discovering aneurysmal formation is short. The term was within a month. In all patients, the papillary muscle was in the proximity of the orifice of the aneurysm. Patch repair of these unusual left ventricular aneurysms may be effective for improvement of left ventricular function without mitral regurgitation.

Dual involvement of coenzyme Q10 in redox signaling and inhibition of death signaling in the rat heart mitochondria.

Coenzyme Q10 (CoQ) has long been utilized as a cardioprotective agent in various heart diseases. One of the most important mechanisms by which CoQ exerts cardioprotection is aerobic ATP production as a mobile electron carrier in the mitochondrial electron transfer chain. The ability of CoQ to afford myocardial protection is also attributed to its antioxidant property. However, CoQ may also act as a pro-oxidant through the generation of reactive oxygen species. Although excess oxidative stress is known to induce death signaling via cytochrome c release from mitochondria, it is now apparent that a brief exposure to oxidative stress stimulates redox signaling for acquisition of tolerance to oxidative stress. Therefore, we have investigated dual involvement of CoQ in redox signaling generation through enhanced production of reactive oxygen species and death signaling inhibition through antioxidation. Mitochondria were isolated from the rat heart and incubated with CoQ (10 or 100 microM) or its vehicle HCO 60 for 1 h. H2O2 and cytochrome c release from respiring mitochondria were increased by antimycin A (2 microM), an inhibitor of complex III respiratory chain, or by high Ca2+ (10 microM). This enhanced release of H2O2 was associated with an increase in lipid peroxidation as measured with 4-hydroxy-2-nonenal-modified proteins and with large amplitude swelling of mitochondria. CoQ potentiated H2O2 release from antimycin A- or high Ca(2+)-treated mitochondria, but was capable of inhibiting lipid peroxidation and large amplitude swelling, and attenuated cytochrome c release from the mitochondria. In addition, CoQ increased ATP synthesis by mitochondria. These results suggest that CoQ plays dual roles in mitochondrial generation of intracellular signaling. CoQ acts as a pro-oxidant that participates in redox signaling. CoQ also acts as an antioxidant that inhibits permeability transition and cytochrome c release, and increases ATP synthesis, thereby attenuating death signaling toward apoptosis and necrosis.

IGF-I differentially regulates Bcl-xL and Bax and confers myocardial protection in the rat heart.

Bcl-2 family proteins play a crucial role in the cytoprotective action of insulin-like growth factor-I (IGF-I) by regulating cell death signaling at the mitochondrial level. The present study examined the effect of IGF-I on the expression of Bcl-2 family proteins in the rat heart mitochondria in relation to myocardial protection against ischemia-reperfusion injury. Systemic IGF-I (1 mg) treatment in the rat increased Bcl-xL and attenuated Bax 12-24 h later in the heart mitochondria fraction. Permeability transition and cytochrome c release occurred in a Ca(2+) concentration-dependent manner in the vehicle-treated mitochondria. This was significantly inhibited by the IGF-I-pretreatment. Moreover, ATP synthesis was significantly greater in the IGF-I-pretreated mitochondria. IGF-I pretreatment 24 h before 25 min of global ischemia in the isolated rat heart model significantly improved recovery of isovolumic left ventricular function and inhibited creatine kinase release during reperfusion. This was associated with a significantly less number of terminal transferase labeling-positive myocytes and nonmyocytes 2 h after reperfusion. These results suggest that IGF-1 differentially regulates Bcl-xL and Bax in heart mitochondria, which may be causally related to myocardial protection against ischemia-reperfusion injury.

Protein kinase C isoform-dependent myocardial protection by ischemic preconditioning and potassium cardioplegia.

OBJECTIVE: Ischemic preconditioning combined with potassium cardioplegia does not always confer additive myocardial protection. This study tested the hypothesis that the efficacy of ischemic preconditioning under potassium cardioplegia is dependent on protein kinase C isoform. METHODS: Isolated and crystalloid-perfused rat hearts underwent 5 cycles of 1 minute of ischemia and 5 minutes of reperfusion (low-grade ischemic preconditioning) or 3 cycles of 5 minutes of ischemia and 5 minutes of reperfusion (high-grade ischemic preconditioning) or time-matched continuous perfusion. These hearts received a further 5 minutes of infusion of normal buffer or oxygenated potassium cardioplegic solution. The isoform nonselective protein kinase C inhibitor chelerythrine (5 micromol/L) was administered throughout the preischemic period. All hearts underwent 35 minutes of normothermic global ischemia followed by 30 minutes of reperfusion. Isovolumic left ventricular function and creatine kinase release were measured as the end points of myocardial protection. Distribution of protein kinase C alpha, delta, and epsilon in the cytosol and the membrane fractions were analyzed by Western blotting and quantified by a densitometric assay. RESULTS: Low-grade ischemic preconditioning was almost as beneficial as potassium cardioplegia in improving functional recovery; left ventricular developed pressure 30 minutes after reperfusion was 70 +/- 15 mm Hg (P <.01) in low-grade ischemic preconditioning and 77 +/- 14 mm Hg (P <.001) in potassium cardioplegia compared with values found in unprotected control hearts (39 +/- 12 mm Hg). Creatine kinase release during reperfusion was also equally inhibited by low-grade ischemic preconditioning (18.2 +/- 10.6 IU/g dry weight, P <.05) and potassium cardioplegia (17.6 +/- 6.7 IU/g, P <.01) compared with control values. However, low-grade ischemic preconditioning in combination with potassium cardioplegia conferred no significant additional myocardial protection; left ventricular developed pressure was 80 +/- 17 mm Hg, and creatine kinase release was 14.8 +/- 11.0 IU/g. In contrast, high-grade ischemic preconditioning with potassium cardioplegia conferred better myocardial protection than potassium cardioplegia alone; left ventricular developed pressure was 121 +/- 16 mm Hg (P <.001), and creatine kinase release was 8.3 +/- 5.8 IU/g (P <.05). Chelerythrine itself had no significant effect on functional recovery and creatine kinase release in the control hearts, but it did inhibit the salutary effects not only of low-grade and high-grade ischemic preconditioning but also those of potassium cardioplegia. Low-grade ischemic preconditioning and potassium cardioplegia enhanced translocation of protein kinase C alpha to the membrane, whereas high-grade ischemic preconditioning also enhanced translocation of protein kinase C delta and epsilon. Chelerythrine inhibited translocation of all 3 protein kinase C isoforms. CONCLUSIONS: These results suggest that myocardial protection by low-grade ischemic preconditioning and potassium cardioplegia are mediated through enhanced translocation of protein kinase C alpha to the membrane. It is therefore suggested that activation of the novel protein kinase C isoforms is necessary to potentiate myocardial protection under potassium cardioplegia.