Mathematical modeling and quality parameters of Salicornia fruticosa dried by convective drying.

The effect of convective drying at 50, 60 and 70 °C on the drying kinetics and quality parameters of Salicornia fruticosa was investigated. To estimate the equilibrium moisture content a desorption isotherm was performed using five empirical models: Halsey, Caurie, Henderson, Smith and Oswin. The experimental data was also fitted to different drying kinetic models (Logarithmic, Two-Terms, Midilli-Kucuk and Exponential Two-Terms). A numerical simulation using the Finite Volume Method allowed us to describe the evolution of temperature and moisture content distributions during drying. The Henderson model was found to be the most suitable for predicting the equilibrium moisture content of S. fruticosa, with values of X we in the drying process of 1.51; 1.54 and 1.36 g water/g d.m for 50, 60 and 70 °C, respectively. A good agreement was found between the numerical and experimental results of temperature and moisture during Salicornia drying. The Midilli-Kucuk model presented the best fitting to the drying curves. The effects of drying on S. fruticosa were significant in two quality parameters. Antioxidant capacity decreased in ca. 45% and lightness (> L*) significantly increased at a drying temperature of 70 °C, compared to the fresh samples. The optimum drying temperature where drying time and nutrients loss was minimum was 70 °C. These results can be used to estimate the best drying conditions for producing dehydrated Salicornia. The use of halophytes as sustainable crops is promising, and the vision of their commercial production must be evaluated and considered, given water scarcity in many areas of the planet.

When the earth trembles in the Americas: the experience of Haiti and Chile 2010.

The response of the nephrological community to the Haiti and Chile earthquakes which occurred in the first months of 2010 is described. In Haiti, renal support was organized by the Renal Disaster Relief Task Force (RDRTF) of the International Society of Nephrology (ISN) in close collaboration with Médecins Sans Frontières (MSF), and covered both patients with acute kidney injury (AKI) and patients with chronic kidney disease (CKD). The majority of AKI patients (19/27) suffered from crush syndrome and recovered their kidney function. The remaining 8 patients with AKI showed acute-to-chronic renal failure with very low recovery rates. The intervention of the RDRTF-ISN involved 25 volunteers of 9 nationalities, lasted exactly 2 months, and was characterized by major organizational difficulties and problems to create awareness among other rescue teams regarding the availability of dialysis possibilities. Part of the Haitian patients with AKI reached the Dominican Republic (DR) and received their therapy there. The nephrological community in the DR was able to cope with this extra patient load. In both Haiti and the DR, dialysis treatment was able to be prevented in at least 40 patients by screening and adequate fluid administration. Since laboratory facilities were destroyed in Port-au-Prince and were thus lacking during the first weeks of the intervention, the use from the very beginning on of a point-of-care device (i-STAT®) was very efficient for the detection of aberrant kidney function and electrolyte parameters. In Chile, nephrological problems were essentially related to difficulties delivering dialysis treatment to CKD patients, due to the damage to several units. This necessitated the reallocation of patients and the adaptation of their schedules. The problems could be handled by the local nephrologists. These observations illustrate that local and international preparedness might be life-saving if renal problems occur in earthquake circumstances.

Renal L-type fatty acid-binding protein mediates the bezafibrate reduction of cisplatin-induced acute kidney injury.

Fibrates, the PPAR alpha ligand-like compounds increase the expression of proximal tubule liver fatty acid binding protein (L-FABP) and significantly decrease cisplatin-induced acute kidney injury. To study whether the bezafibrate-mediated upregulation of renal L-FABP was involved in this cytoprotective effect we treated transgenic mice of PPAR agonists inducible human L-FABP expression with cisplatin in the presence or absence of bezafibrate. Blood urea nitrogen was unchanged in the first day but increased 3 days after cisplatin. While urinary L-FABP increased over 100-fold 1 day after cisplatin treatment in the transgenic mice it was significantly reduced when these transgenic mice were pretreated with bezafibrate. Cisplatin-induced renal necrosis and apoptosis were significantly reduced in bezafibrate pretreated transgenic mice and this correlated with decreased accumulation of lipid and lipid peroxidation products. Immunohistochemical analysis of kidney tissue of bezafibrate-cisplatin-treated transgenic mice showed preservation of cytoplasmic L-FABP in the proximal tubule, but this was reduced in transgenic mice treated only with cisplatin. L-FABP mRNA and protein levels were significantly increased in bezafibrate-cisplatin-treated transgenic mice when compared to mice not fibrate treated. Our study shows that the bezafibrate-mediated upregulation of proximal tubule L-FABP plays a pivotal role in the reduction of cisplatin-induced acute kidney injury.

Liver fatty acid-binding protein as a biomarker of acute kidney injury after cardiac surgery.

Acute kidney injury (AKI) is a major complication of cardiac bypass surgery. We examined whether levels of liver fatty acid-binding protein (L-FABP) can be an early biomarker for ischemic injury by measuring this protein in the urine of 40 pediatric patients prior to and following cardiopulmonary bypass surgery. AKI was defined as a 50% increase in the serum creatinine from baseline, which was normally not seen until 24-72 h after surgery. Enzyme-linked immunosorbent assay analysis showed increased L-FABP levels (factored for creatinine excretion) of about 94- and 45-fold at 4 and 12 h, respectively, following surgery in the 21 patients who developed AKI with western blot analysis, confirming L-FABP identity. Univariate logistic regression analyses showed that both bypass time and urinary L-FABP were significant independent risk indicators for AKI. After excluding bypass time from the model and using a stepwise multivariate logistic regression analysis, urinary L-FABP levels at 4 h after surgery were an independent risk indicator with the area under the receiver-operating characteristic curve 0.810, sensitivity 0.714, and specificity 0.684 for a 24-fold increase in urinary L-FABP. Our study shows that urinary L-FABP levels represent a sensitive and predictive early biomarker of AKI after cardiac surgery.

A role of liver fatty acid-binding protein in cisplatin-induced acute renal failure.

Previous studies from our laboratory showed that increased fatty acid oxidation by the kidney is cytoprotective during cisplatin (CP)-mediated nephrotoxicity. In this study, we determined the effects of CP and fibrates on peroxisome proliferation and the expression of liver fatty acid-binding protein (L-FABP) in normal mice, and in mice transgenically overexpressing human L-FABP (h-L-FABP). Labeling of peroxisomes demonstrated reduced peroxisomal staining in the proximal tubule of CP-treated mice compared with control mice. There was increased peroxisomal labeling in the proximal tubules of both control and CP-treated mice when either was treated with fibrate; a known peroxisome proliferator-activated receptor-alpha ligand. L-FABP protein expression, not detected in control or CP-treated mice, was significantly increased in the proximal tubules of fibrate-treated mice of either group. In the transgenic mice, CP increased the shedding of h-L-FABP in the urine, which was decreased by fibrate as was the acute renal failure. A cytosolic pattern of h-L-FABP expression was found in the proximal tubules of untreated transgenic mice with a nuclear presence in CP-treated mice. Fibrate pretreatment restored the cytosolic expression pattern in CP-treated mice. Our study shows that fibrate may improve CP-induced acute renal failure due to both peroxisome proliferation and increased L-FABP in the cytosol of the proximal tubule.

Metabolomic study of cisplatin-induced nephrotoxicity.

We have shown that cisplatin inhibits fatty acid oxidation, and that fibrate treatment ameliorates renal function by preventing the inhibition of fatty acid oxidation and proximal tubule cell death. Urine samples of mice treated with single injection of cisplatin (20 mg/kg body weight) were collected for 3 days and analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy. In a separate group, urine samples of mice treated with peroxisome proliferator-activated receptor-alpha (PPARalpha) ligand WY were also analyzed by NMR after 2 days of cisplatin exposure. Biochemical analysis of endogenous metabolites was performed in serum, urine, and kidney tissue. Electron microscopic studies were carried out to examine the effects of PPARalpha ligand and cisplatin. Principal component analysis demonstrated the presence of glucose, amino acids, and trichloacetic acid cycle metabolites in the urine after 48 h of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also demonstrated the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, in spite of increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of PPARalpha ligand. Electron microscopic analysis confirmed the protective effect of the fibrate on preventing cisplatin-mediated necrosis of the S3 segment of the proximal tubule. Our study shows that cisplatin-induces a unique NMR metabolic profile in urine of mice that developed acute renal failure, and confirms the protective effect of a fibrate class of PPARalpha ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity.

Etomoxir-induced PPARalpha-modulated enzymes protect during acute renal failure.

Regulation of fatty acid beta-oxidation (FAO) represents an important mechanism for a sustained balance of energy production/utilization in kidney tissue. To examine the role of stimulated FAO during ischemia, Etomoxir (Eto), clofibrate, and WY-14,643 compounds were given 5 days prior to the induction of ischemia/reperfusion (I/R) injury. Compared with rats administered vehicle, Eto-, clofibrate-, and WY-treated rats had lower blood urea nitrogen and serum creatinines following I/R injury. Histological analysis confirmed a significant amelioration of acute tubular necrosis. I/R injury led to a threefold reduction of mRNA and protein levels of acyl CoA oxidase (AOX) and cytochrome P4A1, as well as twofold inhibition of their enzymatic activities. Eto treatment prevented the reduction of mRNA and protein levels and the inhibition of the enzymatic activities of these two peroxisome proliferator-activated receptor-alpha (PPARalpha) target genes during I/R injury. PPARalpha null mice subjected to I/R injury demonstrated significantly enhanced cortical necrosis and worse kidney function compared with wild-type controls. These results suggest that upregulation of PPARalpha-modulated FAO genes has an important role in the observed cytoprotection during I/R injury.

Role of fatty acid beta-oxidation and calcium-independent phospholipase A2 in ischemic acute renal failure.

Membrane phospholipolysis during ischemic cell injury is accompanied by the activation of a novel calcium-independent phospholipase A2 in the proximal tubule. Long-chain fatty acid metabolic products produced by phospholipase A2 activation accumulate during ischemia as a result of the inhibition of fatty acid beta-oxidation on the mitochondria and peroxisomes. Altogether, lysophospholipids, long-chain acyl carnitines, and long-chain acyl coenzyme A inhibit proximal tubule Na+K(+)-ATPase. Metabolic regulation of the gene expression of fatty acid beta-oxidation enzymes during ischemic acute renal failure may represent a novel therapeutic maneuver to enhance the recovery of kidney function during ischemia.

cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities.

Previous studies have suggested that activation of calcium-independent PLA2 (CaIPLA2) is an early event in cell death after hypoxic injury in proximal tubule cells. An approximately 28-kD CaIPLA2 with preferential activity toward plasmalogen phospholipids has been recently purified from rabbit kidney cortex (D. Portilla and G. Dai, J Biol Chem 271, 15,451-15,457, 1996). Their report describes the cloning of a full-length rat cDNA encoding CaIPLA2, using sequences derived from the purified rabbit kidney cortex enzyme. In addition, cDNA from rabbit kidney that encode the rabbit homologue of the enzyme and a closely related isoform were isolated. The rat cDNA is predicted to encode an approximately 24-kD protein, and each cDNA contains the sequence G-F-S-Q-G, which fits the active site consensus sequence G-X-S-X-G of carboxylesterases. Several lines of evidence (DNA sequence comparison, Southern blot analysis, and examination of the expressed sequence tag database) show that CaIPLA2 enzymes are encoded by a multigene family in rats, mice, rabbits, and humans. Northern analysis of various tissues from the rat indicated that the CaIPLA2 gene is ubiquitously expressed, with highest mRNA abundance observed in the kidney and small intestine. The rat CaIPLA2 cDNA, when expressed in a baculovirus expression system, and the purified rabbit kidney cortex protein exhibit both CaIPLA2 and lysophospholipase activities. The cloned CaIPLA2 cDNA are expected to aid in understanding the role of CaIPLA2 in cell death after hypoxic/ischemic cell injury.

Carnitine palmitoyl-transferase enzyme inhibition protects proximal tubules during hypoxia.

The role of inhibition of the CPT enzymes responsible for accumulation of long chain acylcarnitines (LCAC) during hypoxia in the proximal tubule has not been previously examined. We have characterized CPT enzyme activities in mitochondrial fractions of rabbit proximal tubules. Malonyl CoA-sensitive CPT I activity (1.1 +/- 0.3 nmol/min/mg protein), and detergent-solubilized, malonyl CoA-insensitive CPT II activity (2.3 +/- 0.4 nmol/min/mg protein) were readily detected in proximal tubule mitochondrial fractions. Subjecting rabbit proximal tubules to various periods of hypoxia did not significantly change mitochondrial CPT I or CPT II activities. Thirty minutes of hypoxia resulted in an increase in lysophospholipid mass from 440 +/- 105 to 720 +/- 93 pmol/mg protein, N = 5, LCAC mass from 79 +/- 11 to 618 +/- 34 pmol/mg protein, N = 5, and lactate dehydrogenase (LDH) release from 9 +/- 1% to 46 +/- 3%, N = 8. Pretreatment of proximal tubules with two different CPT inhibitors, glybenclamide (Glyb) 400 microM and oxfenicine (Oxfe) 1 mM, resulted in reduction in the magnitude of hypoxia-induced lysophospholipid formation 490 +/- 160 (Glyb), 342 +/- 150 pmol/mg protein (Oxfe), N = 4, hypoxia-induced LCAC formation 295 +/- 27 (Glyb), 128 +/- 16 pmol/mg protein (Oxfe). N = 5, and LDH release 25 +/- 1% (Glyb) and 19 +/- 2% (Oxfe), N = 8. The protective effect of CPT inhibition was also associated with increased production of lactate suggesting the modulation of a substrate-mediated metabolic switch. Immunoblots demonstrated that hypoxia caused a time dependent hydrolysis of fodrin-alpha subunit and that CPT inhibition protected against hypoxia-induced fodrin proteolysis. These data suggest a unifying hypothesis that links phospholipase A2 (PLA2) activation, and hypoxia-mediated fodrin proteolysis to the proximal tubule mitochondrial CPT system. I propose that CPT inhibition may represent a novel mechanism to ameliorate proximal tubule cell death during hypoxia.

Purification of a novel calcium-independent phospholipase A2 from rabbit kidney.

We have recently identified a cytosolic calcium-independent phospholipase A2 (PLA2) that represents the major measurable PLA2 activity in rabbit proximal tubules (Portilla, D., Shah, S. V., Lehman, P. A., and Creer, M. H.(1994) J. Clin. Invest. 93, 1609-1615). We now report the 3200-fold purification of this PLA2 to homogeneity from rabbit kidney cortex through sequential column chromatography including anion exchange, hydrophobic interaction, Mono Q, hydroxylapatite, phenyl-Sepharose, and chromatofocusing fast protein liquid chromatography from rabbit kidney cortex. The purified enzyme had a molecular mass of 28 kDa, possessed a specific activity of 1.2 micronol/mg min and a neutral pH optimum, and exhibited a preferential hydrolysis toward sn-2 fatty acid from diradylglycerophospholipids. The purified polypeptide hydrolyzed plasmenylcholine > phosphatidylcholine glycerophospholipids and selectively cleaved phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic acid. Antibodies against the purified protein precipitated all of the soluble calcium-independent PLA2 activity from rabbit kidney cortex. These data altogether suggest that the 28-kDa protein in the kidney represents a novel class of calcium-independent PLA2.

Hypoxia-induced amphiphiles inhibit renal Na+, K(+)-ATPase.

We have characterized the effects of hypoxia on carnitine metabolism in proximal tubules. Hypoxia for 10 minutes resulted in a significant increase in the mass of long chain acylcarnitines (LCAC) (control 53 +/- 20 vs. hypoxia 118 +/- 38 pmol. mg-1 protein). Since LCAC are proximal metabolites in the beta-oxidation of fatty acids, these data suggest that inhibition of fatty acid oxidation occurs during hypoxia in the proximal tubule. In addition to LCAC accumulation, hypoxia resulted in a significant increase in the mass of lysoplasmenylcholine LPLasCho (control 62 +/- 15 pmol/mg vs. 20 min hypoxia 146 +/- 21 pmol/mg protein, N = 4) and also in increases in the mass of monoacyl LPC (control 122 +/- 24 pmol/mg protein vs. 283 +/- 35 pmol/mg protein after 40 min of hypoxia). We tested the possibility that these compounds that accumulate during hypoxia could inhibit proximal tubule Na+, K(+)-ATPase. LPC, LPlasC, and LCAC inhibited proximal tubule nystatin-stimulated oxygen consumption (QO2) and proximal tubule Na+, K(+)-ATPase activity in a dose-dependent manner. In addition, LPC, LPlasC, and LCAC directly inhibited' (65%, 80%, and 60%, respectively) Na+, K(+)-ATPase activity purified from kidney cortex at similar concentrations at which they accumulate during hypoxia (above 25 microM). The present data suggest that amphiphile accumulation may have a potential pathophysiologic role in the proximal tubule during renal ischemia.

Plasmalogen phospholipid hydrolysis during hypoxic injury of rabbit proximal tubules.

We have identified and quantified the major species of arachidonate-containing phospholipids in proximal tubules by high performance liquid chromatographic and gas chromatographic analyses. Arachidonate was found to comprise 53% of the total mass of fatty acids esterified at the sn-2 position of ethanolamine phospholipids, and 51% of that amount resides in three plasmenylethanolamine species containing the vinyl ethers of palmitaldehyde, oleylaldehyde or stearylaldehyde at the sn-1 position. Choline phospholipids contained 21% arachidonylated species and 33% of that amount resides in a single plasmenylcholine species containing the vinyl ether of palmitaldehyde at the sn-1 position. Ten minutes of hypoxia did not cause a significant change in the total phospholipid mass of ethanolamine or choline phospholipids; however, phosphate analysis of the individual phospholipid molecular species containing esterified arachidonic acid in isolated proximal tubules demonstrated a 24% reduction in the mass of the plasmenylethanolamine molecular species with the vinyl ether of oleylaldehyde at the sn-1 position and a 35% reduction in the mass of plasmenylcholine species with palmitaldehyde at the sn-1 position. These studies underscore the pathophysiological importance of plasmalogen phospholipid hydrolysis and suggest that activation of PLA2s, which utilize endogenous proximal tubule plasmalogen substrates, may play an important role in the early generation of arachidonic acid and accompanying phospholipid catabolism during hypoxic cell injury.

Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules.

Although the activation of calcium-independent phospholipase A2 (PLA2) enzymes has been described in the heart, the pathogenetic role of this enzyme(s) in hypoxic cell injury has not been previously examined in any tissue. Therefore, we characterized the time course of activation of calcium-independent PLA2 using both plasmalogen and diacylglycerophospholipid substrates during hypoxia in rabbit proximal tubules and examined whether inhibition of calcium-independent PLA2 activity is associated with a cytoprotective effect. Subjecting rabbit proximal tubules to hypoxia for 5 min resulted in at least a threefold increase in cytosolic calcium-independent PLA2, which was selective for plasmalogen substrates (control 444 +/- 69 vs hypoxia 1,675 +/- 194 pmol.mg protein-1.min-1, n = 5). In contrast, no changes in PLA2 activity were observed in the presence of 4 mM EGTA in the membrane fraction using plasmenylcholine substrates. 20 min of hypoxia resulted in an increase in arachidonate from 3 +/- 1 to 28 +/- 4 ng/mg protein and lactate dehydrogenase release from 7.5 +/- 2% to 38 +/- 5%, n = 4. Pretreatment of proximal tubules with 10 microM Compound I, a specific inhibitor of calcium-independent PLA2, resulted in reduction in the magnitude of both hypoxia-induced arachidonic acid release (11 +/- 3 ng/mg protein) and lactate dehydrogenase release (18 +/- 4%). Our data indicate that a significant fraction of PLA2 activity in the proximal tubule is calcium-independent and selective for plasmalogen substrates. Furthermore, the activation of this enzyme plays an important role in the pathogenesis of membrane injury during hypoxia in the proximal tubule.

Different guanosine triphosphate-binding proteins couple vasopressin receptor to phospholipase C and phospholipase A2 in glomerular mesangial cells.

To evaluate the identity of the guanosine triphosphate--binding proteins coupling arginine vasopressin receptor occupancy with activation of phospholipase C, leading to Ca2+ mobilization, and activation of phospholipase A2, leading to arachidonate release and prostanoid formation, we used intact cells, saponin-permeabilized cells, and membranes of the rat mesangial cell. Arginine vasopressin 10(-7) mol/L produced a dose-dependent increase in cytosolic Ca2+ to maximal levels of 500 nmol/L with peak responses occurring within 10 seconds of addition of arginine vasopressin to cells in suspension. Arginine vasopressin 10(-7) mol/L elicited a maximal response. These increases were associated temporarily with a fourfold increase in tritiated D-myo-inositol 1,4,5-trisphosphate formation in prelabeled cells. Pertussis toxin (200 ng/ml) did not inhibit the Ca2+ increase nor did it inhibit the increase in tritiated D-myo-inositol 1,4,5-trisphosphate formation, suggesting a pertussis toxin--insensitive signaling pathway for phospholipase C hydrolysis in response to vasopressin. Membranes prepared from mesangial cells increased D-myo-inositol 1,4,5-trisphosphate formation in vitro in response to arginine vasopressin and guanosine-5'-0(3- thiotrisphosphate), and this stimulation was inhibited by guanosine-5'-0(2-thiodiphosphate), confirming the involvement of a guanosine triphosphate--binding protein. In contrast arginine vasopressin stimulated arachidonate release from intact mesangial cells, and this effect was blocked by pretreating cells with pertussis toxin. To demonstrate that this was through a pertussis toxin--sensitive guanosine triphosphate--binding protein, we permeabilized cells with saponin and determined that arginine vasopressin and guanosine-5'-0(3-thiotriphosphate) stimulated the release of arachidonic acid and the stimulation of guanosine-5'-0(3-thiotriphosphate) was inhibited by guanosine-5'-0(2-thiodiphosphate). Finally, pertussis toxin was able to stimulate adenosine diphosphate ribosylation in vivo of a substrate protein in mesangial cell membranes of 41 kd, and this ribosylation was inhibited by pretreating cells with pertussis toxin. These data suggest that the release of arachidonic acid by vasopressin in glomerular mesangial cells is linked to a pertussis toxin--sensitive guanosine triphosphate--binding protein and that this activation of phospholipase C in vasopressin is linked to a pertussis toxin--insensitive guanosine triphosphate--binding protein.

Anoxia induces phospholipase A2 activation in rabbit renal proximal tubules.

Phospholipase A2 (PLA2) activation during anoxic cell injury was determined by use of a variety of approaches in rabbit proximal renal tubules. Arachidonic acid (AA) mass release increased from 4 +/- 1 (normoxia control) to 40 +/- 6 ng/mg protein after 20 min and 106 +/- 16 ng/mg protein after 40 min of anoxia. PLA2 activity was measured by estimating the amount of sn-2 fatty acid released from either 14C-labeled Escherichia coli membranes or [14C]phosphatidylethanolamine (PE) micelles incubated with membrane and cytosolic fractions obtained from normoxic or anoxic tubules. At pH 7.4 and 1 mM Ca, PLA2 activity increased in the 20-min anoxic membrane fractions from 8.1 +/- 2.3 (normoxic) to 15.2 +/- 2.1 pmol.min-1.mg protein-1 (anoxic). When the proximal tubules were homogenized in the absence of Ca, the anoxia-induced PLA2 activity was found to be soluble. Preincubation with pancreatic PLA2 antibody inhibited 50% of both basal and anoxia-stimulated PLA2 activity. Two protein bands (40- and 21-kDa species) immunoreactive to PLA2 antibody were detected in the membrane fraction. A sixfold increase in the immunoreactivity of the 40-kDa band was detected after 40 min of anoxia of proximal tubules. These results suggest that anoxia induces an intracellular PLA2 activity in kidney cells that could be immunologically related to pancreatic PLA2.

Peptide hormones, cytosolic calcium and renal epithelial response.

We have reviewed the evidence that a number of hormones interact with renal tubular epithelial cells. The evidence suggests that in the mammalian renal tubule bradykinin and parathyroid hormone interact with cell surface receptors to initiate the hydrolysis of PIP2 leading to the formation of I 1,4,5P3 and diacylglycerol in the distal and proximal tubule, respectively. The activation of this second messenger system leads to the mobilization of Ca2+ from intracellular stores. Vasopressin does not activate this second messenger system in mammalian renal epithelial cells, and we cannot demonstrate I 1,4,5P3 formation and Ca2+ mobilization either in the rabbit papillary collecting tubules or in MDCK cells. There is evidence emerging, but not discussed here, that angiotensin II may also mediate some of its effects on the mammalian proximal tubule via the inositol polyphosphate second messenger system.

Bradykinin-activated membrane-associated phospholipase C in Madin-Darby canine kidney cells.

Previous studies have demonstrated that bradykinin stimulates the rapid release of inositol 1,4,5 trisphosphate (IP3) from membrane phosphatidylinositol 4,5 bisphosphate (PIP2) in Madin-Darby canine kidney (MDCK) cells. Since current evidence would suggest that the activation of phospholipase C (PLC) is mediated through a guanine nucleotide-binding protein in receptor-mediated activation of PLC, we evaluated the role of guanine nucleotide proteins in receptor-mediated (bradykinin-stimulated) activation of PLC in MDCK cells. Bradykinin at 10(-7) M produced a marked increase in IP3 formation within 10 s increasing from a basal level of 46.2 to 686.6 pmol/mg cell protein a 15-fold increase. Pretreatment of MDCK cells in culture with 200 ng/ml of pertussis toxin for 4 h reduced the bradykinin-stimulated response to 205.8 pmol/mg protein. A 41-kD protein substrate in MDCK membranes was ADP ribosylated in vitro in the presence of pertussis toxin. The ADP ribosylation in vitro was inhibited by pretreatment of the cells in culture with pertussis toxin. Membranes from MDCK cells incubated in the presence of [3H]PIP2/phosphatidyl ethanolamine liposomes demonstrated hydrolysis of [3H]PIP2 with release of [3H]IP3 when GTP 100 microM or GTP gamma S 10 microM was added. Bradykinin 10(-7) M added with GTP 100 microM markedly increased the rate of hydrolysis within 10 s, thus demonstrating a similar time course of PLC activation as intact cells. These results demonstrate that bradykinin binds to its receptor and activates a membrane-associated PLC through a pertussis toxin-sensitive, guanine nucleotide protein.

Protein kinase C modulates phospholipase C and increases arachidonic acid release in bradykinin stimulated MDCK cells.

The tumor promoter phorbol ester (PMA) has been shown to stimulate protein kinase C (PKC) in MDCK cells. At the concentrations that produce stimulation of PKC, PMA (100 microM) inhibits BK-induced I1,4,5P3 (IP3) formation and calcium transients in these cells. 1-5-isoquinolinyl-2-methyl-piperazine (H7) a known inhibitor of PKC in MDCK cells reverses the effect of PMA on BK-stimulated IP3 formation and Ca2+ transients in these cells. PMA also stimulates arachidonate release which can be inhibited by preincubation with H7. A dual mechanism of regulation by PKC at the level of phospholipase C (down regulation) and phospholipase A2 (stimulation) is suggested in these cells.