We describe a simplified method for the isolation of large numbers of nephron segments from rat and rabbit kidneys. In contrast to most previous protocols, the kidneys are not perfused. After removal from the animal, the kidney is sliced and torn in pieces that are subsequently digested in culture medium containing 0.5 mg/ml of collagenase at 37°C. If the preparation is agitated only very gently and infrequently, then the tissue gradually falls apart into a suspension containing long nephron fragments, often consisting of multiple connected segments. These are easily sorted into homogeneous segment populations that can be used for enzyme assays, protein extraction for immunoblotting, and RNA extraction for reverse transcription-polymerase chain reaction, all of which have been done successfully in our laboratory. For comparison, we have also examined cortical collecting tubule segments and cells prepared by the more rigorous protocol described previously (E. Schlatter, U. Fröbe, and R. Greger. Pflügers Arch. 421: 381–387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells maintain their normal architecture and a distinct separation of apical and basolateral membranes.
Accumulation of amyloid beta peptide (Aβ) in the brain is a pathological hallmark of Alzheimer’s disease (AD); the underlying mechanism, however, is not well understood. In this study, we show that expression of plasminogen activator inhibitor 1 (PAI-1), a physiological inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), increases with age in the brain of wild type and Aβ precursor protein-presenilin 1 (APP/PS1) transgenic mice as well as in AD patients. Most importantly, we show that knocking out the PAI-1 gene dramatically reduces Aβ burden in the brain of APP/PS1 mice but has no effect on the levels of full-length APP, alpha or beta C-terminal fragments. Furthermore, we show that knocking out the PAI-1 gene leads to increases in the activities of tPA and plasmin, and the plasmin activity inversely correlates with the amounts of SDS insoluble Aβ40 and Aβ42. Together, these data suggest that increased PAI-1 expression/activity contributes importantly to Aβ accumulation during aging and in AD probably by inhibiting plasminogen activation and thus Aβ degradation.
The voltage-dependent K (KV) channel in Daudi human B lymphoma cells was characterized by using patch-clamp techniques. Whole-cell voltage-clamp experiments demonstrated that cell membrane depolarization induced a transient (time-dependent) outward current followed by a steady-state (time-independent) component. The time-dependent current resembled behavior of the type n channel, such as use dependence and a unique blockade by tetraethylammonium (TEA). Both time-dependent and time-independent currents were blocked by quinine with a similar IC50 (14.2 mM and 12.6 mM). Treatment with antisense oligonucleotide of human Kv1.3 gene significantly reduced both currents by 80%. Single-channel experiments showed that only one type of KV channel was recorded with a unitary conductance of approximately 19 pS. Consistent with whole-cell recordings, the channel activity in cell-attached patches remained in response to prolonged depolarization, and the remaining channel activity was blocked by quinine, but not TEA. Channel activity was scarcely seen in cell-attached patches after antisense treatment. Whole-cell current-clamp data showed that TEA, which blocks only the time-dependent current, caused a slight decrease in the membrane potential. In contrast, quinine and antisense, which block both time-dependent and -independent currents, strongly reduced the membrane potential. These data together suggest that the KV channel in Daudi cells does not completely inactivate and that the remaining channel activity due to this incomplete inactivation appears to be primarily responsible for maintaining the membrane potential.
AVP not only increases osmotic water permeability (Pf) in the rat cortical collecting duct (CCD), but also acts synergistically with aldosterone to augment sodium reabsorption (JNa). These effects are inhibited by catecholamines via alpha2 adrenergic receptors, and by dopamine. We review here studies designed to determine the mechanism and receptor involved in dopamine action. The inhibitory effect of dopamine on Na+ and water transport was found to be reversible, and was not produced by agonists specific to D1A and D1B receptors. D2-type (D2, D3 or D4) receptors and activation of the GTP-binding protein Gi were implicated by the observation that dopamine had no inhibitory effect when JNa and Pf were stimulated by a cyclic AMP analogue plus isobutylmethylxanthine. The only dopaminergic antagonist that reversed the inhibitory effect of dopamine was clozapine, which is relatively D4-specific. We also found that dopamine or D1-specific agonists by themselves had no effect on cAMP production. However, dopamine inhibited the high rate of AVP-dependent cAMP production, and this effect of dopamine was reversed by clozapine but not other antagonists or by inhibitors of protein kinase C. The D4 receptor was observed in western blots of renal cortical proteins, and it was localized to the collecting duct by RT-PCR and immuno-histochemistry using a D4-specific antibody. These results show that at least a portion of the natriuretic effect of dopamine can be attributed to inhibition of AVP-dependent Na+ reabsorption by the CCD, and they introduce another signalling system as a candidate in the aetiology of low-renin, salt-dependent hypertension.
Key words: Alzheimer's disease, isoflavones, I -/B-oestradiol, oestrogen receptor, tau phosphorylation. Abbreviations used: AD, Alzheimer's disease; EGF, epidermal growth factor; ER oestrogen receptor; HME, human mammary epithelial; PTK, protein tyrosine kinase; TGFB, transforming growth factor / I . 'To whom correspondence should be addressed (e-mail helenkrn@uab.edu). placement therapy that is commonly prescribed for women) was not correlated. T h e results discussed here indicate that genistein, and probably other related phytoestrogens, have pleiotropic actions, some of which may involve TGFP activity.
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