Hubert K. Zajicek

SRBC/cavin-3 is a caveolin adapter protein that regulates caveolae function

Caveolae are a major membrane domain common to most cells. One of the defining features of this domain is the protein caveolin. The exact function of caveolin, however, is not clear. One possible function is to attract adapter molecules to caveolae in a manner similar to how clathrin attracts molecules to coated pits. Here, we characterize a candidate adapter molecule called SRBC. SRBC binds PKCδ and is a member of the STICK (substrates that interact with C‐kinase) superfamily of PKC‐binding proteins. We also show it co‐immunoprecipitates with caveolin‐1. A leucine zipper in SRBC is essential for both co‐precipitation with caveolin and localization to caveolae. SRBC remains associated with caveolin when caveolae bud to form vesicles (cavicles) that travel on microtubules to different regions of the cell. In the absence of SRBC, intracellular cavicle traffic is markedly impaired. We conclude that SRBC (sdr‐related gene product that binds to c‐kinase) and two other family members [PTRF (Pol I and transcription release factor) and SDPR] function as caveolin adapter molecules that regulate caveolae function.

Rapid downregulation of rat renal Na/Pi cotransporter in response to parathyroid hormone involves microtubule rearrangement

Renal proximal tubule cells express in their apical brush border membrane (BBM) a Na/P(i) cotransporter type IIa that is rapidly downregulated in response to parathyroid hormone (PTH). We used the rat renal Na/P(i) cotransporter type IIa (NaPi-2) as an in vivo model to assess early cellular events in the rapid downregulation of this transporter. When rats were treated with PTH for 15 minutes, NaPi-2 abundance in the BBM was decreased. In parallel, transporter accumulated in intracellular vesicles. Concomitantly, microtubules (MTs) were found to form dense bundles of apical-to-basal orientation. After 60 minutes of PTH action, the cells were vastly depleted of NaPi-2, whereas their microtubular cytoskeleton had returned to its normal appearance. Prevention of MT rearrangement by taxol resulted in accumulation of NaPi-2 in the subapical cell portion after 15 minutes and a strong delay in depletion of intracellular transporter after 60 minutes of PTH action. Furthermore, the subapical accumulation of NaPi-2 was associated with the expansion of dense apical tubules of the subapical endocytic apparatus (SEA). Depolymerization of MTs by colchicine likewise caused a retardation of intracellular NaPi-2 depletion. These results suggest that NaPi-2 is downregulated in response to PTH through a rapid endocytic process in 2 separate steps: (a) internalization of the transporter into the SEA, and (b) its delivery to degradative organelles by a trafficking mechanism whose efficiency depends on a taxol-sensitive rearrangement of MTs.

Metabolic acidosis regulates rat renal Na-Si cotransport activity

Recently, we cloned a cDNA (NaSi-1) localized to rat renal proximal tubules and encoding the brush-border membrane (BBM) Na gradient-dependent inorganic sulfate (Si) transport protein (Na-Si cotransporter). The purpose of the present study was to determine the effect of metabolic acidosis (MA) on Na-Si cotransport activity and NaSi-1 protein and mRNA expression. In rats with MA for 24 h (but not 6 or 12 h), there was a significant increase in the fractional excretion of Si, which was associated with a 2.4-fold decrease in BBM Na-Sicotransport activity. The decrease in Na-Si cotransport correlated with a 2.8-fold decrease in BBM NaSi-1 protein abundance and a 2.2-fold decrease in cortical NaSi-1 mRNA abundance. The inhibitory effect of MA on BBM Na-Si cotransport was also sustained in rats with chronic (10 days) MA. In addition, in Xenopus laevis oocytes injected with mRNA from kidney cortex, there was a significant reduction in the induced Na-Si cotransport in rats with MA compared with control rats, suggesting that MA causes a decrease in the abundance of functional mRNA encoding the NaSi-1 cotransporter. These findings indicate that MA reduces Si reabsorption by causing decreases in BBM Na-Si cotransport activity and that decreases in the expression of NaSi-1 protein and mRNA abundance, at least in part, play an important role in the inhibition of Na-Si cotransport activity during MA.

Epidermal growth factor inhibits Na-Pi cotransport in weaned and suckling rats

In the present study, we determined the effect of epidermal growth factor (EGF; 10 microgram/100 g body wt) on sodium gradient-dependent phosphate transport (Na-Pi cotransport) regulation in suckling (12-day-old) and weaned (24-day-old) rats. Weaned rats had higher proximal tubular brush border membrane vesicle (BBMV) Na-Pi cotransport activity (232 +/- 16 in weaned vs. 130 +/- 9 pmol. 10 s-1. mg protein-1 in suckling rats, P < 0.05). Chronic treatment with EGF induced inhibition of BBMV Na-Pi cotransport in both suckling (130 +/- 9 vs. 104 +/- 7 pmol. 10 s-1. mg protein-1, P < 0. 05) and weaned rats (232 +/- 16 vs. 145 +/- 9 pmol. 10 s-1. mg protein-1, P < 0.005). The inhibitory effect was selective for Na-Pi cotransport as there was no inhibition of Na-glucose cotransport. Weaned rats had a higher abundance of BBMV NaPi-2 protein than suckling rats (increase of 54%, P < 0.001) and a twofold increase in NaPi-2 mRNA. The EGF-induced inhibition of Na-Pi transport was paralleled by decreases in NaPi-2 protein abundance in both weaned (decrease of 26%, P < 0.01) and suckling (decrease of 27%, P < 0.01) animals. In contrast, there were no changes in NaPi-2 mRNA abundance. We conclude that proximal tubule BBMV Na-Pi cotransport activity, NaPi-2 protein abundance, and NaPi-2 mRNA abundance are higher in weaned than in suckling rats. EGF inhibits Na-Pi cotransport activity in BBMV isolated from suckling and weaned rats, and this inhibition is mediated via a decrease in NaPi-2 protein abundance, in the absence of a change in NaPi-2 mRNA.

Effect of Rocaltrol on Bone Mass in Patients with Pulmonary Disease Treated with Corticosteroids

The aim of this study was to evaluate the effect of calcitriol on bone mass in patients with corticosteroid induced osteoporosis. Thirty-seven patients (26 females, 11 males, mean age 66.4 years) with pulmonary disease under long-term treatment with corticosteroids (5–10 mg prednisolone daily) and osteopenia/osteoporosis verified by dual-energy x-ray absorptiometry (DEXA) measurement were enrolled into the study. Rocaltrol was prescribed to 30/37 of the patients, the rest of the patients (6 females, 1 male) served as controls. In the treatment group, there was a slight increase of bone mass in the hip and lumbar vertebrae (L1–L4), whereas the control group showed a decrease of bone mass (change rate of bone mass in patients +0.8% and +1.0%, respectively, vs. –1.9% and –0.3%, respectively, in the control group). The preliminary results of our study suggest a beneficial role for the treatment of corticosteroid induced osteoporosis with Rocaltrol, which is well-tolerated by patients and cost-efficient in patient management.

Spectroscopy and microscopy of cells and cell membrane systems.

Publisher Summary This chapter describes the spectroscopy and microscopy of cells and cell membrane systems. Spectroscopy and microscopy studies of cells and cell membranes yield important information about lipid and protein dynamics as well as lipid-protein interactions that play an important role in the regulation of cell function. The chapter also concentrates on apical and basolateral membranes isolated from the kidney to illustrate how studies with these plasma membrane fractions yield important information about differential lipid dynamics in apical versus basolateral membranes, mainly due to differential lipid composition. The bright fluorescence is an interesting property of aorta matrix proteins. Their morphology and their physiological remodeling or pathological modifications, such as bending and rupture, have a fundamental significance in the life of the blood vessel. When imaging vessel matrix proteins, an important reason suggests the use of two-photon excitation instead of the conventional one-photon excitation: the tissue can be observed under physiological conditions, with no need for fixation, labeling, or embedding in matrices and thin sectioning, procedures that may damage the tissue and alter the protein morphology.