Epithelial Ca Research Articles

Synthesis of charybdotoxin and of two N‐terminal truncated analogues

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.

Toxin Sensitivity of the Calcium-Dependent Rubidium Efflux in Madin-Darby Canine Kidney Cells

86Rb+ efflux was measured on polarized Madin-Darby canine kidney cells under A23187 or ATP stimulation. This efflux, inhibited by barium, Leiurus quinquestriatus hebraeus venom and charybdotoxin was attributed to the stimulation of Ca++-activated maxi K+ channels. Snake venom from Dendroaspis polylepis did not alter the stimulation as well as did apamine. ATP was effective on both the apical and basolateral membranes and the Ca++-activated maxi K+ channels were predominently found on the basolateral membrane. This study presents the physiological evidence that dendrotoxin is ineffective on the epithelial Ca++-activated maxi K+ channel present in MDCK cells.

Differential Regulation of Carbonic Anhydrase II by Androgen and Estrogen in Dorsal and Lateral Prostate of the Rat*

Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostate. CA II is a major soluble protein in these accessory sex glands. The immunoelectronmicroscopy showed that CA II is expressed in their epithelial cells only. For studies on hormone regulation, adult male rats were castrated for 2 or 7 days. Groups of 7-day castrates and normal rats were treated daily either with testosterone or 17-beta-estradiol for 6 days and 2-day castrates for 1 day. CA II protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by RIA. The levels of CA II mRNA were studied by Northern blotting and hybridization of total RNA with a 32P-labeled mouse CA II cDNA clone. Castration of the rats decreased the concentration of CA II in lateral prostate but increased in dorsal prostate. These changes were reversed in both prostatic lobes by testosterone treatment. Estrogen treatment of castrated rats enhanced CA II concentration in lateral prostate but no effects were seen in the dorsal prostate of the same animals. In normal rats estrogen increased CA II concentration of dorsal prostate but there was no change in lateral prostate. Corresponding changes were observed in the levels of CA II mRNA in both tissues. The morphometric analyses showed that the castration- and hormone-induced changes of the mRNA and protein levels of the exclusively epithelial CA II could not be explained by any alterations in the proportions of epithelial and stromal components of the glands after hormone manipulations. The results demonstrate the differential steroid regulation of CAII in two prostatic lobes. Androgen regulates the expression of CAII at messenger RNA level, but the responses of CAII to testosterone are opposite in dorsal and lateral prostate. Estrogen increases CA II expression in lateral prostate but in dorsal prostate the castration-like effects of estrogen on CAII expression are probably indirect.

Role of Calcium-Channel Bockers in Preventing Acute and Chronic Renal Injury

Calcium-channel blockers (CCBs) have been shown to afford protection against acute (ARF) and chronic renal failure (CRF). The effects of CCBs against acute renal injury occur at both the vascular and tubular epithelial level. At the vascular level, experimental ARF-associated loss of renal autoregulation and hypersensitivity to renal nerve stimulation has been shown to be reversed by CCBs. These beneficial vascular effects of CCBs occur on the background of the finding that renal ischemic injury is associated with an increase in cellular Ca2+ concentration. A rise in tubular epithelial Ca2+ concentration also occurs very early after a renal ischemic insult. This effect of ischemia is associated with evidence of membrane depolarization, opening of slow calcium channels, increased cellular Ca2+ uptake, and reversal by CCBs. There is evidence that the increased cellular Ca2+ uptake activates phospholipases, which prolong and increase membrane damage. Experimental CRF is also associated with increased renal cellular Ca2+ concentration, an effect that can be attenuated by CCBs. The CCBs probably slow progression of CRF by both cytoprotective and antihypertensive effects. These findings of vascular and tubular effects of CCBs in experimental ARF and CRF have led to their clinical use to prevent initial dysfunction of cadaveric kidney transplants, cyclosporine nephrotoxicity, radiocontrast-induced ARF, and progression of CRF. Randomized clinical studies are necessary to further examine the efficacy of CCBs in ARF and CRF.

Calcium uptake kinetics into brush-border membrane vesicles: Higher Vmax in the spontaneously hypertensive rat than in normotensive control

Arterial hypertension in the spontaneously hypertensive rat (SHR) is associated with an abnormal Ca 2+ homeostasis, compared with its normotensive control, the Wistar Kyoto rat (WKY). In particular, epithelial Ca 2+ transport is perturbed, with intestinal absorption and renal tubular reabsorption being decreased in the adolescent and adult SHR. In the present study we examined Ca 2+ uptake into isolated duodenal brush-border membrane vesicles (BBMV) in 12–14 week-old male rats. This uptake can be separated in a non-saturable and a saturable component. The latter follows Michaelis-Menten kinetics. Vmax of this component was found to be significantly higher (p<0.05) in SHR than in WKY (0.58±0.19 versus 0.35±0.06 nmol/mg protein × 10 sec, mean±SD) whereas Km did not differ. Thus, the defect in the intestinal Ca 2+ absorption previously identified in the SHR of this age is not due to a decrease in Ca uptake at the level of the duodenal brush-border membrane, but is most likely located in the baso-lateral membrane.

Effects of 1,25(OH)2D3 on enterocyte basolateral membrane Ca transport in rats.

One, twenty-five dihydroxyvitamin D3 [1,25(OH)2D3], commonly known as calcitriol, stimulates intestinal Ca absorption through increased activity of a cellular transport process. To determine whether transcellular Ca transport involves energy-dependent Ca efflux across enterocyte plasma membrane in vitamin D-sufficient rats, in vitro bidirectional Ca fluxes were measured under short-circuited conditions across proximal duodenum from rats fed diets adequate in vitamin D and containing a normal Ca diet (NCD), a low Ca diet (LCD), or fed NCD and injected with 50 ng of 1,25(OH)2D3 daily for 4 days before study. LCD or 1,25(OH)2D3 increased Ca net flux [Jnet, mucosal-to-serosal flux minus the serosal-to-mucosal flux] by increasing Ca mucosal-to-serosal flux (Jm----s) (mean +/- SE, NCD vs. LCD vs. 1,25(OH)2D3, 16 +/- 4 vs. 179 +/- 18 vs. 82 +/- 21 nmol.cm-2. h-1, P less than 0.0001). Initial ATP-dependent Ca uptake rates by duodenal basolateral membrane vesicles (BLMV) was greater in vesicles from rats fed NCD compared with LCD and not different from NCD injected with 1,25(OH)2D3. These studies suggest that in vitamin D-replete animals, 1,25(OH)2D3 increases epithelial Ca Jm----s by mechanisms that do not involve ATP-dependent BLM Ca efflux. ATP-dependent Ca exit from the cell under these conditions may play a role in intracellular Ca homeostasis rather than Ca absorption.

Effects of parathyroid hormone on cytosolic free calcium concentration in individual rabbit connecting tubules.

PTH stimulates active Ca reabsorption in isolated perfused rabbit kidney connecting tubules (CNTs). The existence of PTH-sensitive adenylate cyclase and the reproduction of increased epithelial Ca transport by dibutyryl-cAMP suggest that cAMP is the mediator. Accordingly, we studied the effects of PTH and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) on cytosolic free calcium concentration [( Ca2+]i) in individual rabbit CNTs. [Ca2+]i was estimated by continuous epifluorescence microscopy of single fura-2-loaded tubules during dual wave-length excitation. In nonperfused controls at 37 degrees C, [Ca2+]i decreased with time. In contrast to vehicle controls, synthetic bovine (1-34) PTH (0.1 nM) increased [Ca2+]i within 4 min, produced a maximal effect in 7.2 min, and sustained its effect for at least 2 min after washout. 8-Br-cAMP (1 mM) mimicked the effect of PTH, but with an earlier onset of action. To test the hypothesis that lumen Ca is the predominant source of the rise in [Ca2+]i, we studied singly perfused CNTs. In the absence of bath and lumen Ca, PTH elicited no rise in [Ca2+]i, implying that intracellular Ca stores are not the major source. In contrast, there was a rise when Ca was replenished in both media. In the continuous presence of bath Ca, lumen Ca was estimated to contribute 65% of the total rise in [Ca2+]i in response to PTH when it was first deleted and then replenished. However, when the sequence of lumen Ca manipulation was reversed, the contributions by lumen and bath Ca were found to be essentially equal. We conclude (a) at a physiologic concentration, PTH increases [Ca2+]i in rabbit CNTs, (b) 8-Br-cAMP mimics this action, implicating cAMP as a second messenger, and (c) the PTH-stimulated rise in [Ca2+]i depends importantly on both bath and tubular luminal fluid Ca.

Effect of a snake venom cardiotoxin (CTX Bungarus fasciatus) on epithelial Ca 2+-activated K + currents : Qi H. Gong 1, Steven J. Wieland 2, Jeffrey E. Fletcher 1 and Ming S. Jiang1 (Departments of 1Anesthesiology and 2Anatomy, Hahnemann University, Philadelphia, PA 19102, U.S.A.)

Effect of a snake venom cardiotoxin (CTX Bungarus fasciatus) on epithelial Ca 2+-activated K + currents : Qi H. Gong 1, Steven J. Wieland 2, Jeffrey E. Fletcher 1 and Ming S. Jiang1 (Departments of 1Anesthesiology and 2Anatomy, Hahnemann University, Philadelphia, PA 19102, U.S.A.)

Ca2+-ATPase activity in the human lens

A membrane-rich preparation of paired human lenses was prepared in such a manner as to preserve ATPase activity. The lipid:protein ratio of these preparations was increased 12-fold with an 85% recovery of total phospholipid. The pattern of stimulation of ATPase activity by a range of calcium concentrations was found to be similar in membrane preparations of epithelium and cortex. The concentration of calcium necessary for half-maximal simulations of ATPase activity was approximately 10(-6) M. Ca2+-ATPase activity is undetectable in the lens nuclear region. A shift in the sensitivity of lens epithelial Ca2+-ATPase activity was observed with increasing age concomitant with a general increase in Ca2+-ATPase activity suggesting age related modifications of the membrane.

Water flow in the toad urinary bladder in response to vasopressin: role of potassium

In agreement with previous reports, we found that absence of K+ from the serosal bath of the toad urinary bladder substantially impairs vasopressin and cAMP-stimulated water flow. The decreased response to vasopressin appears unrelated to prostaglandin production since inhibition of endogenous prostaglandins by pretreatment with naproxen 10(-5) M failed to prevent the effect seen with K+-free Ringer's. The resistance to vasopressin does not appear to be directly related to epithelial K+ concentrations, in that maneuvers leading to decreased intracellular K+ failed to produce a similar effect. A more likely explanation appears to be that K+-free Ringer's induces an increased cytosolic Ca++ which, in turn, decreases the hydrosmotic effects of vasopressin. Several lines of evidence argue in favor of such an explanation: (a) Increased cytosolic Ca++ had been found in other tissues with low extracellular K+; (b) The resistance to vasopressin decreases with decreased serosal Ca++; (c) The effects of K+-free Ringer's are not additive in situations believed to have increased epithelial Ca++, i.e. replacement of serosal Na+ with choline; (d) The effects of K+-free serosal bathing medium could be both prevented and/or reversed if already established by increasing serosal bath, and presumably intracellular, pH, which is believed to decrease intracellular Ca++.

Water flow in the toad urinary bladder in response to vasopressin: role of potassium

In agreement with previous reports, we found that absence of K+ from the serosal bath of the toad urinary bladder substantially impairs vasopressin and cAMP-stimulated water flow. The decreased response to vasopressin appears unrelated to prostaglandin production since inhibition of endogenous prostaglandins by pretreatment with naproxen 10−5 M failed to prevent the effect seen with K+-free Ringer's. The resistance to vasopressin does not appear to be directly related to epithelial K+ concentrations, in that maneuvers leading to decreased intracellular K+ failed to produce a similar effect. A more likely explanation appears to be that K+-free Ringer's induces an increased cytosolic Ca++ which, in turn, decreases the hydrosmotic effects of vasopressin. Several lines of evidence argue in favor of such an explanation: (a) Increased cytosolic Ca++ had been found in other tissues with low extracellular K+; (b) The resistance to vasopressin decreases with decreased serosal Ca++; (c) The effects of K+-free Ringer's are not additive in situations believed to have increased epithelial Ca++, i.e. replacement of serosal Na+ with choline; (d) The effects of K+-free serosal bathing medium could be both prevented and/or reversed if already established by increasing serosal bath, and presumably intracellular, pH, which is believed to decrease intracellular Ca++.

Relation of fibrin and tumour-associated antigens to the spread of ovarian cancer

Tumour-associated antigens in blood, ascitic fluid and tissue from patients with ovarian cancer were compared with fibrin formation and the occurrence of the fibrinolysis product D-dimer and set into relation to the clinical tumour situation. In 68 patients with epithelial ovarian cancer D-Dimer, CA 125, TPA, CA 19-9 and CEA were investigated as tumour markers. Specificity and sensitivity were compared in sensitivity-specificity diagrams for different clinical tumour situations. Antigen concentrations and incidence of pathological values were related to the extent of the tumour disease. In manifest tumour situations the sensitivity of CA 125 was slightly superior to that of D-dimer at a corresponding specificity level, whereas the sensitivity of the latter was superior in subclinical tumour situations. Immunohistochemical investigations and plasma-ascites diffusion ratios of antigens indicate that enrichment may occur in ascites. The histochemical pattern reflects fibrin as a tumour-surrounding and surface related antigen. Fibrin formation and dissolution seem to be related to the intraabdominal spread of ovarian cancer

Calcium transport across the pars recta of cortical segment 2 proximal tubules.

Partes rectae of cortical segment 2 proximal tubules were dissected from rabbit kidneys and perfused in vitro. Ca concentrations of perfused and collected fluids were measured by continuous-flow microcolorimetry. Epithelial Ca permeability (P) was estimated from the bath-to-lumen movement of 45Ca. The transepithelial voltage (psi) and [Ca2+] difference were varied simultaneously by changing perfusate composition. Tubules that were perfused and bathed with an identical artificial ultrafiltrate of plasma displayed a lumen-negative psi, a collectate [Ca] greater than perfusate, and net Ca secretion. Tubules perfused with "late" proximal tubule fluid (high [Cl], low [HCO3], low concentrations of Na+-cotransported solutes) demonstrated a lumen-positive psi, a perfusate [Ca2+] greater than the bath, a collectate [Ca] less than perfusate, and net Ca absorption. Under each of these conditions, net Ca flux was in the direction predicted by the experimentally measured driving forces for diffusional Ca transport. Tubules that were cooled while being perfused with late proximal tubule fluid showed an increased lumen-positive psi but reduced net Ca absorption. The latter finding was consistent with reduced Ca ion diffusion related to a smaller P at the lower temperature. I conclude that Ca2+ diffusion is an important component of net Ca absorption in this segment of the nephron.

Tumorassoziierte Antigene und Fibrinderivate als Reaktionsprodukte des Ovarialkarzinoms

The detection of tumour-associated antigens in blood, ascites fluid, and tissue by the use of monoclonal antibodies establishes new aspects for the course control of ovarian cancer therapy and the evaluation of tumour-associated fibrinolysis. Monoclonal antibodies to the cross-linking site of fibrin derivatives in an enzyme immunoassay (D-dimer-ELISA) allow to compare quantitative results of fibrinolysis products with different tumour markers and the clinical tumour situation. In a prospective follow-up-study in 102 patients with epithelial ovarian carcinoma Ca 125, D-dimer, TPA, Ca 19-9, and CEA were investigated as tumour markers. Specificity (control group 61 gynaecologic patients with benign diseases) and sensitivity were compared in sensitivity-specificity diagrams (receiver operating characteristic curves) for different clinical tumour situations (preoperative, S0-S3). Applying a limit value of normal range, indicated in parentheses, the specificity of Ca 125 (greater than 65 mu/ml) amounts to 100%, D-dimer (greater than 700 ng/ml) 100%, TPA (greater than 120 mu/ml) 90%, CEA (greater than 5 ng/ml) 96%, Ca 19-9 (greater than 37 mu/ml) 98%. In the same sequence, sensitivity values came to 91%, 82%, 65%, 8% and 19% in manifest tumour situations. Antigen-concentration levels and frequency of pathological values depend on the degree of tumour disease. The results of seroimmunodiagnostic methods correlate with the clinical tumour situation: In 11 of 12 cases clinical tumour progression or relapse were accompanied by increasing concentrations of Ca 125 and D-dimer. In 21 cases of second-look laparotomies, tumour marker values in the normal range were often found not to be conclusive for diagnosing a tumour-free situation. The predictive value for true negative results amounted to only 18%. Immunohistochemical investigations and plasma-ascites diffusion ratios of antigens indicate that enrichment occurs in ascites. Fibrin deposition often surrounds tumour plugs and is found in interstitional tissue, whereas Ca 125 is expressed mainly at tumour cell surface. The histochemical image reflects fibrin as a tumour surrounding antigen and Ca 125 as a tumour-originated antigen. Clinical relevant serodiagnostic methods for ovarian cancer are brought about by the monoclonal detection of the investigated antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Micropuncture study of magnesium transport along the nephron in the young rat.

Micropuncture study of magnesium transport along the nephron in the young rat.

A study of cerebral palsy in the childhood population of Edinburgh.

Vitamin D plays an important role in Ca²⁺ homeostasis by controlling Ca²⁺ (re)absorption in intestine, kidney, and bone. The epithelial Ca²⁺ channel TRPV5 mediates the Ca²⁺ entry step in active Ca²⁺ reabsorption. TRPV5 knockout (TRPV5^−/−) mice show impaired Ca²⁺ reabsorption, hypercalciuria, hypervitaminosis D, and intestinal hyperabsorption of Ca²⁺. Moreover, these mice demonstrate upregulation of intestinal TRPV6 and calbindin-D_9K expression compared with wild-type mice. For addressing the role of the observed hypervitaminosis D in the maintenance of Ca²⁺ homeostasis and the regulation of expression levels of the Ca²⁺ transport proteins in kidney and intestine, TRPV5/25-hydroxyvitamin-D₃-1α-hydroxylase double knockout (TRPV5^−/−/1α-OHase^−/−) mice, which show undetectable serum 1,25(OH)₂D₃ levels, were generated. TRPV5^−/−/1α-OHase^−/− mice displayed a significant hypocalcemia compared with wild-type mice (1.10 ± 0.02 and 2.54 ± 0.01 mM, respectively; <i>P</i> &lt; 0.05). mRNA levels of renal calbindin-D_28K (7 ± 2%), calbindin-D_9K (32 ± 4%), Na⁺/Ca²⁺ exchanger (12 ± 2%), and intestinal TRPV6 (40 ± 8%) and calbindin-D_9K (26 ± 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5^−/−/1α-OHase^−/− mice, more pronounced than observed in single TRPV5 or 1α-OHase knockout mice. It is interesting that a renal Ca²⁺ leak, as demonstrated in TRPV5^−/− mice, persisted in TRPV5^−/−/1α-OHase^−/− mice, but a compensatory upregulation of intestinal Ca²⁺ transporters was abolished. In conclusion, the elevation of serum 1,25(OH)₂D₃ levels in TRPV5^−/− mice is responsible for the upregulation of intestinal Ca²⁺ transporters and Ca²⁺ hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca²⁺ reabsorption in TRPV5^−/− mice.