Role of O-linked N-acetylglucosamine modification in diabetic nephropathy.

Increased O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) is a known contributor to diabetes; however, its relevance in diabetic nephropathy (DN) is poorly elucidated. Here, we studied the process and enzymes of O-GlcNAcylation with a special emphasis on Akt-endothelial nitric oxide synthase (eNOS) and heat shock protein (HSP)72 signaling. Since tubular injury is the prominent site of DN, the effect of hyperglycemia was first measured in proximal tubular (HK2) cells cultured in high glucose. In vivo O-GlcNAcylation and protein levels of O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), phosphorylated (p)Akt/Akt, peNOS/eNOS, and HSP72 were assessed in the kidney cortex of streptozotocin-induced diabetic rats. The effects of various renin-angiotensin-aldosterone system (RAAS) inhibitors were also evaluated. In proximal tubular cells, hyperglycemia-induced OGT expression led to increased O-GlcNAcylation, which was followed by a compensatory increase of OGA. In parallel, peNOS and pAkt levels decreased, whereas HSP72 increased. In diabetic rats, elevated O-GlcNAcylation was accompanied by decreased OGT and OGA. RAAS inhibitors ameliorated diabetes-induced kidney damage and prevented the elevation of O-GlcNAcylation and the decrement of pAkt, peNOS, and HSP72. In conclusion, hyperglycemia-induced elevation of O-GlcNAcylation contributes to the progression of DN via inhibition of Akt/eNOS phosphorylation and HSP72 induction. RAAS blockers successfully inhibit this process, suggesting a novel pathomechanism of their renoprotective action in the treatment of DN.

Aldosterone antagonists in monotherapy are protective against streptozotocin-induced diabetic nephropathy in rats.

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers.

Altered gene expression profiles in the hippocampus and prefrontal cortex of type 2 diabetic rats.

BACKGROUND: There has been an increasing body of epidemiologic and biochemical evidence implying the role of cerebral insulin resistance in Alzheimer-type dementia. For a better understanding of the insulin effect on the central nervous system, we performed microarray-based global gene expression profiling in the hippocampus, striatum and prefrontal cortex of streptozotocin-induced and spontaneously diabetic Goto-Kakizaki rats as model animals for type 1 and type 2 diabetes, respectively. RESULTS: Following pathway analysis and validation of gene lists by real-time polymerase chain reaction, 30 genes from the hippocampus, such as the inhibitory neuropeptide galanin, synuclein gamma and uncoupling protein 2, and 22 genes from the prefrontal cortex, e.g. galanin receptor 2, protein kinase C gamma and epsilon, ABCA1 (ATP-Binding Cassette A1), CD47 (Cluster of Differentiation 47) and the RET (Rearranged During Transfection) protooncogene, were found to exhibit altered expression levels in type 2 diabetic model animals in comparison to non-diabetic control animals. These gene lists proved to be partly overlapping and encompassed genes related to neurotransmission, lipid metabolism, neuronal development, insulin secretion, oxidative damage and DNA repair. On the other hand, no significant alterations were found in the transcriptomes of the corpus striatum in the same animals. Changes in the cerebral gene expression profiles seemed to be specific for the type 2 diabetic model, as no such alterations were found in streptozotocin-treated animals. CONCLUSIONS: According to our knowledge this is the first characterization of the whole-genome expression changes of specific brain regions in a diabetic model. Our findings shed light on the complex role of insulin signaling in fine-tuning brain functions, and provide further experimental evidence in support of the recently elaborated theory of type 3 diabetes.

Renoprotective effect of erythropoietin in rats subjected to ischemia/reperfusion injury: gender differences.

BACKGROUND: Renal ischemia reperfusion injury induces gender-dependent heat-shock protein 72 expression, which maintains membrane localization of renal Na(+)/K(+)ATPase-α1. The erythropoietin has a protecting effect against ischemia reperfusion injury in various organs. In this study, we investigated whether erythropoietin exerts a beneficial effect against post-ischemic renal injury. Furthermore, we studied the erythropoietin signaling on heat-shock protein 72 and Na(+)/K(+)ATPase-α1 expression and localization. METHODS: In male and female Wistar rats, rHuEPO (1000 IU/bwkg intraperitoneal) or vehicle was administered 24 hours prior to unilateral left renal ischemia reperfusion (50 minutes). Kidneys were subsequently removed at hours 2 or 24 of the reperfusion; sham-operated rats served as controls (C) (n = 8/group). We measured serum erythropoietin, renal function, evaluated histological injury, and observed heat-shock protein 72 as well as Na(+)/K(+)ATPase-α1 protein level and localization. Additional groups were followed for 7-day survival. RESULTS: Erythropoietin treatment was associated with better post-ischemic survival and less impaired renal function in males while diminishing the renal structural damage in both sexes. Endogenous erythropoietin was higher in males and increased in both genders after erythropoietin treatment. The erythropoietin treatment elevated protein levels of heat-shock protein 72 and Na(+)/K(+)ATPase-α1 in 24 hours in males, whereas in females, the already higher expression of heat-shock protein 72 and Na(+)/K(+)ATPase-α1 was not increased. Moreover, erythropoietin prevented ischemia reperfusion induced Na(+)/K(+)ATPase-α1 translocation from the basolaterale membrane in males. CONCLUSION: Erythropoietin diminishes gender difference in the susceptibility to renal post-ischemic injury and reduces post-ischemic structural damage while preserving kidney function, particularly in males. This additional protection may be associated with a heat-shock protein 72-mediated effect on Na(+)/K(+)ATPase-α1 expression and translocation.

Insulin induced translocation of Na+/K+ -ATPase is decreased in the heart of streptozotocin diabetic rats.

AIM: To investigate the effect of acute insulin administration on the subcellular localization of Na(+)/K(+)-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats. METHODS: Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na(+)/K(+)-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity. RESULTS: In control rat heart muscle alpha1 isoform of Na(+)/K(+) ATPase resides mainly in the plasma membrane fraction, while alpha2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of alpha1 isoform (25 %, P<0.05) in plasma membrane and alpha2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha2- but not alpha1-subunits was detected. Alpha1-subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of alpha2 increased by 3.3-fold, alpha1 by 1.37-fold and beta1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive. CONCLUSION: This study demonstrates that insulin influences the plasma membrane localization of Na(+)/K(+)-ATPase isoforms in the heart. alpha2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. alpha1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na(+)/K(+)-ATPase alpha1- and alpha2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.

The slow sarco/endoplasmic reticulum Ca2+ -ATPase declines independently of slow myosin in soleus muscle of diabetic rats.

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) isoforms are normally expressed in coordination with the corresponding myosin heavy chain (MyHC) isoforms in the fibers of skeletal muscle but this coordination is often disrupted in pathological conditions. In the streptozotocin-induced diabetes of rats (stz-rats), the soleus muscle showed peripheral neuropathy and the SERCA2a level decreased in type I (slow-oxidative) fibers compared to the control muscles, whereas the expression of the corresponding slow MyHC1 did not change. No difference was found at the mRNA and protein levels of SERCA and MyHC isoforms in the whole soleus, except that the level of the SERCA2a protein specifically declined in stz-rats compared to the controls. This shows that the coordinated expression of SERCA2a and MyHC1 is disrupted at the SERCA2a protein level in the diabetic soleus. The results are in line with previous observations that regulators of the Ca-homeostasis may adapt faster to type I diabetes than the contractile elements.

Association of extracellular superoxide dismutase (SOD3) Ala40Thr gene polymorphism with pre-eclampsia complicated by severe fetal growth restriction.

OBJECTIVE: Placental and systemic oxidative stress with an imbalance in the oxidant/antioxidant activity seems to play a central role in the pathogenesis of pre-eclampsia. The aim of our study was to examine whether two missense polymorphisms of the extracellular superoxide dismutase (SOD3) gene (Arg213Gly and Ala40Thr) are associated with pre-eclampsia in a Caucasian population from Hungary. STUDY DESIGN: One hundred and fifty-nine pre-eclamptic patients and 114 normotensive, healthy pregnant women were involved in this case-control study. The SOD3 Arg213Gly and Ala40Thr genotypes were determined using the polymerase chain reaction-restriction length polymorphism (PCR-RFLP) and allele-specific amplification methods. RESULTS: The Arg213Gly variant was not detected in our population. There were no significant differences in the genotype and allele frequencies of the SOD3 Ala40Thr polymorphism between pre-eclamptic patients and control subjects. However, the mutant allele carriers of this polymorphism showed an increased risk for severe fetal growth restriction-complicated pre-eclampsia, which was independent of maternal age, prepregnancy BMI, primiparity and smoking status (OR: 6.07, 95% CI: 1.33-27.8, p=0.020; adjusted OR: 4.89, 95% CI: 1.03-23.2, p=0.046). CONCLUSION: Our results suggest a role of SOD3 Ala40Thr single nucleotide polymorphism in the risk of severe fetal growth restriction-complicated pre-eclampsia. However, further studies are needed with a larger sample size to confirm our findings and to explore the exact molecular basis of this observation.

Na+,K+-ATPase is modulated by angiotensin II in diabetic rat kidney--another reason for diabetic nephropathy?

Angiotensin II (ANGII) plays a central role in the enhanced sodium reabsorption in early type 1 diabetes in man and in streptozotocin-induced (STZ) diabetic rats. This study investigates the effect of untreated STZ-diabetes leading to diabetic nephropathy in combination with ANGII treatment, on the abundance and localization of the renal Na(+),K(+)-ATPase (NKA), a major contributor of renal sodium handling. After 7 weeks of STZ-diabetes (i.v. 65 mg kg(-1)) a subgroup of control (C) and diabetic (D7) Wistar rats were treated with ANGII (s.c. minipump 33 microg kg(-1) h(-1) for 24 h; CA and D7A). We measured renal function and mRNA expression, protein level, Serin23 phosphorylation, subcellular distribution, and enzyme activity of NKA alpha-1 subunit in the kidney cortex. Diabetes increased serum creatinine and urea nitrogen levels (C versus D7), as did ANGII (C versus CA, D7 versus D7A). Both diabetes (C versus D7) and ANGII increased NKA alpha-1 protein level and enzyme activity (C versus CA, D7 versus D7A). Furthermore, the combination led to an additive increase (D7 versus D7A, CA versus D7A). NKA alpha-1 Ser23 phosphorylation was higher both in D7 and ANGII-treated rats in the non-cytoskeletal fraction, while no signal was detected in the cytoskeletal fraction. Control kidneys showed NKA alpha-1 immunopositivity on the basolateral membrane of proximal tubular cells, while both D7 and ANGII broadened NKA immunopositivity towards the cytoplasm. Our study demonstrates that diabetes mellitus (DM) increases the mRNA expression, protein level, Ser23 phosphorylation and enzyme activity of renal NKA, which is further elevated by ANGII. Despite an increase in total NKA quantity in diabetic nephropathy, the redistribution to the cystosol suggests the Na(+) pump is no longer functional. ANGII also caused translocation from the basolateral membrane, thus in diabetic states where ANGII level is acutely elevated, the loss of NKA will be exacerbated. This provides another mechanism by which ANGII blockade is likely to be protective.

Association between estrogen receptor alpha (ESR1) gene polymorphisms and severe preeclampsia.

Associations have been reported between estrogen receptor alpha (ESR1) gene polymorphisms and various pathological conditions, including cardiovascular diseases. Our aim was to investigate whether two polymorphisms of the ESR1 gene (ESR1 c.454 -397T>C: PvuII restriction site and c.454 -351A>G: XbaI restriction site) are associated with preeclampsia. In a case-control study, we analyzed blood samples from 119 severely preeclamptic patients and 103 normotensive, healthy pregnant women using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. All of the women were Caucasian. There was no association between severe preeclampsia and the PvuII and XbaI ESR1 gene polymorphisms separately. However, with the simultaneous carriage of both polymorphisms, the TT/AA genotype combination was significantly more frequent in severely preeclamptic patients than in healthy control subjects (24.4% vs. 9.7%, p=0.003), whereas the TT/AG combination was significantly less frequent in the severely preeclamptic group than in the control group (5.0% vs. 18.4%, p=0.002). According to the haplotype estimation, the homozygous T-A haplotype carriers had an increased risk of severe preeclampsia independent of maternal age, prepregnancy BMI, primiparity and smoking status (adjusted odds ratio [OR]: 4.36, 95% confidence interval [CI]: 1.65-11.53). The GG genotype of the XbaI polymorphism was associated with a lower risk of fetal growth restriction in patients with severe preeclampsia (OR: 0.23, 95% CI: 0.07-0.73). In conclusion, the homozygous T-A haplotype carriers of ESR1 PvuII and XbaI polymorphisms showed an increased risk of severe preeclampsia. In addition, the GG genotype of the XbaI polymorphism decreased the risk of fetal growth restriction in severely preeclamptic patients.

Changes of the different neuropeptide-containing nerve fibers and immunocells in the diabetic rat's alimentary tract.

Peripheral neuropathy is a common complication of diabetes mellitus, where neuropeptides and immunocells might play important roles in the pathogenesis of the disease. In this article we have quantified the different neuropeptide-containing nerve fibers and immunocells in the streptozotocin-induced diabetic rat's alimentary tract (tongue, duodenum, colon) using immunohistochemical and immunocytochemical methods. The immunoreactive (IR) nerve fibers were found in all layers of the alimentary tract and their distribution pattern was similar in both control and diabetic groups. Mast cell-nerve fiber contacts were rarely found in the controls. However, after 4 weeks duration of diabetes the number of IR nerve fibers and the immunocompetent cells increased significantly (P < 0.05), and the number of mast cell-nerve fiber contacts was even more significantly increased (P < 0.001). The distance between nerve fibers and immunocells was about 1 mum or even less. Some of the mast cells were degranulated in the vicinity of nerve fibers. No immunocompetent cells were IR for any antisera in the control. However, after the streptozotocin treatment, a large number of the immunocompetent cells showed immunoreactivity for SP and NPY. Counting all immunocompetent cells in whole sections showed that 12.3% of them were IR for SP and 25.4% were IR for NPY. Increased number of SP-containing nerve fibers and immunocells in diabetes mellitus might be the reason for painful neuropathy and might amplify the inflammatory reaction in an axon reflex manner; the released histamine and leukotrienes, cytokines, and chemokines might cause inflammations and lesions of the mucosa.

Leptin receptor gene polymorphisms in severely pre-eclamptic women.

Variants of the leptin receptor gene (LEPR) may modulate the effect of elevated serum leptin levels in pre-eclampsia. The aim of our study was to evaluate the LEPR gene polymorphisms Lys109Arg (A109G) and Gln223Arg (A223G) in severely pre-eclamptic women. In a case-control study, we analyzed blood samples from 124 severely pre-eclamptic patients and 107 healthy control women by the polymerase chain reaction-restriction fragment length polymorphism method. The Pearson chi2 test was used to estimate odds ratios (OR) and 95% confidence intervals (CI). The association was adjusted for maternal age, pre-pregnancy body mass index and primiparity with logistic regression analysis. Pregnant women with the LEPR 223G allele (223A/G or 223G/G genotype) had almost double the risk of developing severe pre-eclampsia compared with patients with the 223A/A genotype (adjusted OR = 1.92, 95% CI: 1.07-3.41). Genotype variants of LEPR A109G alone did not affect the risk of severe pre-eclampsia. Haplotype estimation of A109G and A223G polymorphisms of the LEPR gene revealed that the G-A haplotype versus other pooled haplotypes was significantly less common in the pre-eclamptic group (p < 0.01), while the G-G haplotype versus others was overrepresented among severely pre-eclamptic patients (p < 0.01), compared with controls. In conclusion, our data indicate that LEPR A223G polymorphism may individually modify the risk of severe pre-eclampsia.

Sex differences in heat shock protein 72 expression and localization in rats following renal ischemia-reperfusion injury.

Previously, we demonstrated gender differences in Na-K-ATPase (NKA) expression and function after renal ischemia-reperfusion (I/R) injury (Sex differences in the alterations of Na(+), K(+)-ATPase following ischemia-reperfusion injury in the rat kidney. J Physiol 555: 471-480, 2004). Postischemic membrane destruction causes inhibition of NKA, whereas heat shock protein (HSP) 72 helps to preserve it. We tested the sex differences in postischemic expression of HSP72 and colocalization with NKA. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 (T2), 16 (T16), and 24 h (T24) of reperfusion. Uninephrectomized, sham-operated F and M rats served as controls. Postischemic blood urea nitrogen (BUN), serum creatinine, and renal histology were analyzed. HSP72 mRNA expression was detected by RT-PCR, protein levels by Western blot analysis. Fluorescent immunohistochemistry was performed to evaluate the localization of HSP72 and NKA alpha(1)-subunit. Postischemic BUN and creatinine were higher, and renal histology showed more rapid progression in M vs. F (P < 0.05). HSP72 mRNA expression was higher in F vs. M in control and in all I/R groups (P < 0.05). Similar changes were observed in HSP72 protein levels (F vs. M, P < 0.05, control, T2, T16, T24, respectively). Immunohistochemical localization of HSP72 and NKA alpha(1) was similar in control F and M. In postischemic F kidneys, the majority of NKA alpha(1) and HSP72 was colocalized on the basolateral membrane of tubular cells, whereas in M prominent staining was observed in the cytosol and apical domain. This study indicates that in female kidneys the higher basal and postischemic levels of HSP72 and different colocalization with NKA might contribute to the gender differences in renal I/R injury.

Is preeclampsia associated with higher frequency of HSP70 gene polymorphisms?

AIM: To evaluate the possible association of three different HSP70 gene polymorphisms with preeclampsia. STUDY DESIGN: HSPA1A G(190)C, HSPA1B A(1267)G and HSPA1L T(2437)C polymorphisms were analyzed from blood samples of 72 women with preeclampsia and of 70 healthy pregnant women as controls by PCR-RFLP method. RESULTS: HSPA1B (1267)GG and HSPA1L (2437)CC genotypes occurred more frequently in preeclamptic patients compared to healthy controls (p<0.002 [RR: 4.38, 95% CI: 1.56-12.28]) and (p<0.03 [RR: 1.31, 95% CI: 1.03-1.67]), respectively. Significant difference was found in the distribution of HSPA1B A(1267)G genotype between the preeclamptic and control group (p<0.004 [RR: 0.67, 95% CI: 0.51-0.88]). Distribution of HSPA1A G(190)C was similar in the preeclamptic and control group. In controls, genotype distribution of HSPA1A G(190)C and HSPA1L T(2437)C was in Hardy-Weinberg equilibrium, while this criterion was not fulfilled for HSPA1B A(1267)G. CONCLUSION: We concluded that HSPA1B (1267)GG and HSPA1L (2437)CC genotypes were more frequent among preeclamptic than control patients, suggesting that these genotypes may play a role in the susceptibility for preeclampsia.

Insulin treatment decreases the antioxidant defense mechanism in experimental diabetes.

BACKGROUND: Hyperglycemia-induced oxidative stress and left ventricular hypertrophy is an independent risk factor for cardiovascular disease. Our aim was to determine the relationship between left ventricular hypertrophy and the total scavenger capacity (TSC) in diabetic rats. MATERIAL/METHODS: Control animals (n=30) were compared to streptozotocin-induced diabetic rats (n=38) and diabe-tic rats receiving insulin treatment (n=30) for 22 days. Half of the animals received angiotensin II on day 21. The left ventricular mass, TSC and carbohydrate parameters were determined. RESULTS: Diabetes did not alter left ventricular mass; however, diabetes with insulin treatment was associated with left ventricular hypertrophy. The extent of ventricular hypertrophy did not change significantly in this group after angiotensin II treatment. TSC increased very significantly in diabetic rats. These differences remained high when angiotensin II-treated control rats [CA] were compared with treated diabetic rats [DA] (p<0.005). TSC decreased significantly when diabetic rats were treated with insulin. CONCLUSIONS: Diabetes results in oxidative stress, which in turn enhances the activity of antioxidant enzymes. This increase in enzyme activity inhibits the subcellular remodeling processes, thus also inhibiting concomitant cardiac hypertrophy. Insulin treatment decreases the activity of the antioxidant system and can enhance the function of other localized tissue-specific growth factors. These changes may contribute to the early onset of cardiovascular damage detected in diabetic patients.

Plasticity of the different neuropeptide-containing nerve fibres in the tongue of the diabetic rat.

Common oral complications of diabetes mellitus are xerostomia, impairment of taste, atrophic lesions of the tongue, leukoplakia, lichen oris planus, and tumours, which might be the consequence of chronic inflammation and changes in innervation. In this work, we examined the density of different neuropeptide-containing nerve fibres immunohisto- and immunocytochemically in the root of the control and diabetic rat's tongue. Quantitative analysis showed that the number of immunoreactive (IR) nerve fibres was decreased after 1 week of the streptozotocin treatment, which was prevented by immediate insulin treatment. However, after 4 weeks duration of diabetes, the number of all investigated IR nerve fibres increased significantly (p<0.05), which was further enhanced by the delayed insulin treatment. The numbers of substance P (SP) and vasoactive intestinal polypeptide IR perikarya were also increased by insulin treatment. The electron-microscopic investigations showed that some of the nerve terminals from diabetic animals were found in degeneration. After 4 weeks duration of diabetes, the number of inflammatory cells as well as the mast cell/nerve fibre contacts was also increased. The immunocells also showed IR for SP and neuropeptide Y in the diabetic rats. The insulin treatment decreased both the number and the immunoreactivity of these cells. The increased synthesis and/or regeneration of neuropeptide-containing nerves might indicate the plasticity of nerve fibres in diabetes mellitus, which might happen as a consequence of the changes in the level of neurotrophic factors released by increased number of inflammatory cells or as an effect of insulin.

[New steroid hormone family: endogenous cardiac glycosides and their role in physiologic and pathologic conditions]. / Uj szteroidhormon-család: az endogén szívglikozidok és szerepük a szervezetben fiziológiás és patológiás körülmények között.

In the last two decades extensive study has been carried out on the isolation, identification and biosynthesis of the "endogenous digitalis-like compounds" whose physiological and pathophysiological functions are only starting to be understood. Besides ouabain (strophanthin) and digoxin, four further endogenous cardiac glycosides were isolated and identified so far. These compounds are found in almost all mammalian tissues, including blood plasma and urine, but with the highest concentrations in the adrenal gland, pituitary and hypothalamus. De novo biosynthesis of these glycosides occurs in zona fasciculata cells of adrenal glands, precursors such as progesterone, pregnenolone, and rhamnose increase the synthesis of the ouabain-like immunoreactive material. The secretion of these compounds from the adrenocortical cells are controlled by adrenerg mechanisms, as well as via the renin-angiotensin system. The hydrophobic cardiac glycosides are transported in blood as complexes bound to specific binding globulins. The identified endogenous cardiac glycosides fulfill all the postulated criterions of the hormones, so they represent a new class of steroid hormones. The cardiac glycosides influence the active sodium pump, indirectly the intracellular free calcium concentration and therefore exert a positive inotropic effect on cardiac muscle. Furthermore, in physiological concentrations they can regulate the cell growth and protein synthesis inducing activation of intracellular signal pathways. Under pathological conditions, however, when the concentration of these steroids are high, they play a crucial role in the development of different serious illnesses such as essential hypertension as well as congestive heart failure. Further intensive investigations are needed to clarify some contradictory details accumulated during the last few years in this field.

Sex differences in the alterations of Na(+), K(+)-ATPase following ischaemia-reperfusion injury in the rat kidney.

Postischaemic acute renal failure (ARF) is influenced by sex. Na(+), K(+)-ATPase (NKA) plays a crucial role in the pathogenesis of postischaemic ARF. We tested the impact of sex on mRNA, protein expression, cellular distribution and enzyme activity of NKA following renal ischaemia-reperfusion (I-R) injury. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 h (T2) and 16 h (T16) of reperfusion. Uninephrectomized, sham-operated F and M rats served as controls (n= 6 per group). Blood urea nitrogen, serum creatinine and renal histology were evaluated to detect the severity of postischaemic ARF. mRNA expression of NKA alpha1 and beta1 subunits were detected by RT-PCR. The effect of I-R on cellular distribution was compared by Triton X-100 extraction. Cellular proteins were divided into Triton-insoluble and Triton-soluble fractions and assessed by Western blot. NKA enzyme activity was also determined. After the ischaemic insult blood urea nitrogen and serum creatinine were higher and renal histology showed more rapid progression in M versus F (P < 0.05). mRNA expression of the NKA alpha1 subunit decreased in I-R groups versus controls, but was higher in F versus M both in control and I-R groups (P < 0.05). However, protein levels of the NKA alpha1 subunit in total tissue homogenate did not differ in controls, but were higher in F versus M in I-R groups (P < 0.05). Triton X-100 extractability was lower in F versus M at T16 (P < 0.05). NKA enzyme activity was the same in controls, but was higher in F versus M in I-R groups (T2: 14.9 +/- 2.3 versus 9.15 +/- 2.21 U) (T16: 11.7 +/- 4.1 versus 5.65 +/- 2.3 U; P < 0.05). mRNA and protein expression of the NKA beta1 subunit did not differ between F and M in any of the protocol. We concluded that NKA is more protected from the detrimental effects of postischaemic injury in females. Higher mRNA and protein expression of the NKA alpha1 subunit and higher enzyme activity might be additional contributing factors to the improved postischaemic renal function of female rats.

Molecular forms of acetylcholinesterase in the rat extensor digitorum longus and soleus muscles regenerating from notexin-induced necrosis.

The activity of acetylcholinesterase molecular forms were examined after separation on sucrose gradients during notexin-induced necrosis and the following regeneration in rat extensor digitorum longus (EDL) and soleus (SOL) muscles. All forms dropped rapidly in both muscles in the first few days after single notexin injection. After a delay small globular forms (G1+G2) started to regenerate from day 7 and larger forms (G4 and A12) from day 10 in EDL. The A8 form which cannot be detected in normal EDL was present between day 7 and day 28. In SOL the recovery of AChE forms begun already on day 3. The small globular forms displayed a more rapid increase between day 3 and day 7 then the other forms. In SOL we observed a temporary overshooting peak at day 7 in the activity of all molecular forms. Both muscles recovered their normal AChE pattern by that time when muscle fibres regained their normal diameter (day 28). Most of the events of regeneration of AChE forms resembled those of normal myogenesis.