Smooth muscle calcium and endothelium-derived relaxing factor in the abnormal vascular responses of acute renal failure.

Abnormal renovascular reactivity, characterized by paradoxical vasoconstriction to a reduction in renal perfusion pressure (RPP) in the autoregulatory range, increased sensitivity to renal nerve stimulation (RNS), and loss of vasodilatation to acetylcholine have all been demonstrated in ischemic acute renal failure (ARF). To determine if ischemic injury alters vascular contractility by increasing smooth muscle cell calcium or calcium influx, the renal blood flow (RBF) response to reductions in RPP within the autoregulatory range and to RNS were tested before and after a 90-min intrarenal infusion of verapamil or diltiazem in 7-d ischemic ARF rats. Both calcium entry blockers, verapamil and diltiazem, blocked the aberrant vasoconstrictor response to a reduction in RPP and RNS (both P less than 0.001). In a second series of experiments the potential role of an ischemia-induced endothelial injury and of the absence of endothelium-derived relaxing factor (EDRF) production were examined to explain the lack of vasodilatation to acetylcholine. Acetylcholine, bradykinin (a second EDRF-dependent vasodilator), or prostacyclin, an EDRF-independent vasodilator, was infused intrarenally for 90 min, and RBF responses to a reduction in RPP and RNS were tested in 7-d ischemic ARF rats. Neither acetylcholine nor bradykinin caused vasodilatation or altered the slope of the relationship between RBF and RPP. By contrast, prostacyclin increased RBF (P less than 0.001), but did not change the vascular response to changes in RPP. It was concluded that the abnormal pressor sensitivity to a reduction in RPP and RNS was due to changes in renovascular smooth muscle cell calcium activity that could be blocked by calcium entry blockers. A lack of response to EDRF-dependent vasodilators, as a result of ischemic endothelial injury, may contribute to the increased pressor sensitivity of the renal vessels.     

In vitro activity of Ro 23-6240, a new fluorinated 4-quinolone.

The in vitro activity of Ro 23-6240 (AM833), 6,8-difluoro-1-(2-fluoroethyl)-1,4-dihydro-4-oxo-7(4-methyl-1-piper azinyl) quinolone-3-carboxylic acid, was compared with those of norfloxacin, enoxacin, ofloxacin, and ciprofloxacin. Ro 23-6240 inhibited the majority of Enterobacteriaceae isolates at a concentration of less than or equal to 0.5 microgram/ml. It was especially active against Shigella sp., Salmonella sp., Escherichia coli, and Yersinia enterocolitica, with an MIC for 90% of the strains of less than or equal to 0.12 microgram/ml. The MIC for 90% of the strains was 1 microgram/ml for Serratia marcescens and 8 micrograms/ml for Pseudomonas aeruginosa. Staphylococcus aureus isolates, including methicillin-resistant strains, were inhibited by less than or equal to 1 microgram/ml. Streptococcal and anaerobic species were inhibited by 8 to 16 micrograms/ml. Ro 23-6240 inhibited beta-lactamase-producing bacteria resistant to broad-spectrum cephalosporins. The overall activity of Ro 23-6240 was similar to those of enoxacin, norfloxacin, and ofloxacin, but less than that of ciprofloxacin. The frequency of spontaneous resistance was low, although resistant bacteria could be isolated by repeated subculture. The activity of Ro 23-6240 was decreased in the presence of magnesium at concentrations similar to those present in urine.     

Retrograde perfusion as a model for testing the relative effects of glucose versus insulin on the A cell.

In order to determine whether the A cell may be directly suppressed by glucose in the absence of insulin, canine pancreata were perfused in vitro, both antegrade, through the arterial system and retrograde, through the venous system. Studies of the islet microvasculature have suggested that blood flows from the B cell core to the mantle; thus, the A cell may be tonically inhibited by intra-islet insulin. Retrograde perfusion may then be expected to prevent insulin from reaching the A cell, releasing it from inhibition. Retrograde perfusion with 88 mg/dl glucose markedly increased both insulin and glucagon secretion relative to antegrade levels. In a series of experiments, glucose concentrations were changed from 88 to 200 mg/dl. An antegrade glucose change resulted in increased insulin (134+/-21%; P less than 0.0025) and decreased glucagon (-26+/-9%, P less than 0.025) secretion. A retrograde glucose increase resulted in increased secretion of both insulin (91+/-15%; P less than 0.0005) and glucagon (23+/-9%; P less than 0.0125). To confirm that retrograde perfusion deprived the A cell of endogenous core derived, vascularly delivered insulin, possibly resulting in increased insulin sensitivity, 0.3 mU/ml exogenous porcine insulin was infused. Antegrade, 0.3 mU/ml insulin, had no effect on glucagon secretion (P less than 0.250), while retrograde infusion of 0.3 mU/ml insulin significantly inhibited glucagon secretion (-31 + 8%; P less than 0.0005). The results of our study support the concept that the direction of blood flow and of flow-dependent intra-islet hormone interactions are from the islet B cell core to the mantle. It was further concluded that the normal A cell may not be suppressed by glucose in the absence of insulin.     

In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin angiotensin system.

We examined angiotensinogen gene expression in rat kidney by in situ hybridization histochemistry. Using a rat cRNA probe to angiotensinogen, we demonstrated angiotensinogen mRNA to be localized predominantly in the proximal renal tubule, with considerably lesser amounts in distal tubular segments and glomerular tufts. Previous studies have localized renin immunoreactivity to the juxtaglomerular cells, glomerular tufts, and proximal tubules. Such findings provide further evidence for a local tissue renin angiotensin system within the kidney which may influence regional function. Based on our data, we hypothesize that a major site of angiotensin production is the proximal tubule. We postulate that angiotensin synthesized in and/or around the proximal tubule may directly modulate tubular transport of sodium, bicarbonate, and water. In addition to the proximal tubule, the specific localization of the renin angiotensin components elsewhere in the kidney would also support the other proposed regional functions of the intrarenal system, including modulation of tubuloglomerular balance.     

Evidence of a novel staphylococcal mec-encoded element (mecR) controlling expression of penicillin-binding protein 2''.

A region was identified on the methicillin resistance determinant (mec) isolated from Staphylococcus epidermidis and cloned into Staphylococcus carnosus which was responsible for a novel downregulation of the expression of methicillin resistance. The presence of this region reduced the overall expression of methicillin resistance and the synthesis of the mec-encoded penicillin-binding protein 2' (PBP 2') in S. carnosus. This region was located by Bal31 deletion mutagenesis upstream of the structural gene for PBP 2'. Deletions within this region resulted in higher levels of expression of methicillin resistance and increased levels of PBP 2' synthesis. We tentatively called this region mecR. Analysis of selected Mcr strains of Staphylococcus aureus and S. epidermidis by Southern hybridization suggested that the natural occurrence of two types of mec resistance determinants differ by the presence or absence of mecR-specific sequences.     

Preconditioning rabbit cardiomyocytes: role of pH, vacuolar proton ATPase, and apoptosis.

Ischemic preconditioning signals through protein kinase C (PKC) to protect against myocardial infarction. This protection is characterized by diminished intracellular acidification. Acidification is also a feature of apoptosis, and several agents act to prevent apoptosis by preventing acidification through activation of ion channels and pumps to promote cytoplasmic alkalinization. We characterized metabolic inhibition, recovery, and preconditioning through a PKC-dependent pathway in cardiomyocytes isolated from adult rabbit hearts. Preconditioning reduced loss of viability assessed by morphology and reduced DNA nicking. Blockade of the vacuolar proton ATPase (VPATPase) prevented the effect of preconditioning to reduce metabolic inhibition-induced acidosis, loss of viability, and DNA nicking. The beneficial effect of Na+/H+ exchange inhibition, which is thought to be effective through reduced intracellular Na+ and Ca++, was also abrogated by VPATPase blockade, suggesting that acidification even in the absence of Na+/H+ exchange may lead to cell death. We conclude that a target of PKC in mediating preconditioning is activation of the VPATPase with resultant attenuation of intracellular acidification during metabolic inhibition. Inhibition of the "death protease," interleukin-1-beta converting enzyme or related enzymes, also protected against the injury that followed metabolic inhibition. This observation, coupled with the detection of DNA nicking in cells subjected to metabolic inhibition, suggests that apoptotic cell death may be preventable in this model of ischemia/reperfusion injury.     

Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients.

To examine the mechanism of resistance to fluoroquinolones in Mycobacterium tuberculosis, we selected spontaneous fluoroquinolone-resistant mutants from a susceptible strain, H37Rv, and studied the susceptibilities of these mutants and two fluoroquinolone-resistant clinical isolates (A-382, A-564) to various fluoroquinolones and to isoniazid and rifampin. Furthermore, since mutations within the quinolone resistance-determining region of the structural gene encoding the A subunit of DNA gyrase are the most common mechanism of acquired resistance, we amplified this region by PCR and compared the nucleotide sequences of the fluoroquinolone-resistant strains with that of the susceptible strain. Fluoroquinolone-resistant mutants of H37Rv appeared at frequencies of 2 x 10(-6) to 1 x 10(-8). For three mutants selected on ciprofloxacin, ofloxacin, and sparfloxacin, respectively, and the two clinical isolates, MICs of ciprofloxacin and ofloxacin were as high as 16 micrograms/ml, and those of sparfloxacin were 4 to 8 micrograms/ml. They displayed cross-resistance to all fluoroquinolones tested but not to isoniazid or rifampin. Sparfloxacin and FQ-A (PD 127391-0002) were the most potent fluoroquinolones. All of the fluoroquinolone-resistant strains (MICs, > or = 4 micrograms/ml) had mutations in the quinolone resistance-determining region which led to substitution of the Asp residue at position 87 (Asp-87) by Asn or Ala or the substitution of Ala-83 by Val in the A subunit of DNA gyrase. Similar mutations have been noted in other bacterial species and recently in mycobacteria. The broad resistance to fluoroquinolones that arose readily by point mutation in the laboratory and apparently during inadequate therapy, as was the case in the clinical isolates, may ultimately lead to to serious restriction of the use of these drugs in the treatment of tuberculosis.     

Modulation of skeletal muscle sodium channels by human myotonin protein kinase.

In myotonic muscular dystrophy, abnormal muscle Na currents underlie myotonic discharges. Since the myotonic muscular dystrophy gene encodes a product, human myotonin protein kinase, with structural similarity to protein kinases, we tested the idea that human myotonin protein kinase modulates skeletal muscle Na channels. Coexpression of human myotonin protein kinase with rat skeletal muscle Na channels in Xenopus oocytes reduced the amplitude of Na currents and accelerated current decay. The effect required the presence of a potential phosphorylation site in the inactivation mechanism of the channel. The mutation responsible for human disease, trinucleotide repeats in the 3' untranslated region, did not prevent the effect. The consequence of an abnormal amount of the kinase would be altered muscle cell excitability, consistent with the clinical finding of myotonia in myotonic dystrophy.     

Effects of polyaspartic acid on pharmacokinetics of tobramycin in two strains of rat.

To provide insight into polyaspartic acid nephroprotection and differences in aminoglycoside renal toxicity between two rat strains, the single-dose pharmacokinetics of tobramycin was examined in the presence and absence of polyaspartic acid. Following a single subcutaneous 6.5-mg/kg dose of tobramycin alone, higher aminoglycoside concentrations were measured in Sprague-Dawley rats than in Fischer rats (P < 0.05). Simultaneous administration of polyaspartic acid (50 mg/kg) and tobramycin did not alter the concentrations of tobramycin in serum. The amount of tobramycin in renal tissue and the amount recovered in urine over a 24-h period were greater in both rat strains when tobramycin and polyaspartic acid were given concomitantly. In summary, polyaspartic acid did not alter the concentrations in serum achieved after a single dose of tobramycin in two different rat strains but did result in higher renal concentrations and greater urinary excretion of tobramycin.     

Guanine nucleotide-binding protein, alpha i-3, directly activates a cation channel in rat renal inner medullary collecting duct cells.

We examined whether GTP binding proteins (G proteins) regulate sodium conducting channels in the apical membrane of renal inner medullary collecting duct (IMCD) cells and thereby modulate sodium absorption. Patch clamp studies were conducted on inside-out patches of the apical membrane of IMCD cells grown in primary culture. Guanosine 5'-triphosphate (GTP) and the nonhydrolyzable GTP analogue, GTP gamma S, which activate G proteins, increased the open probability of the cation channel. In contrast, the nonhydrolyzable GDP analogue, GDP beta S, which decreases G protein activity, inhibited the channel. Pertussis toxin also reduced the open probability of the channel. Addition of the alpha *i-3 subunit of Gi to the solution bathing the cytoplasmic surface of the membrane increased the open probability in a dose-dependent manner (2-200 pM). The threshold concentration for activation by alpha *i-3 was 2 pM. Activation of the cation channel by alpha *i-3 was not mediated via a protein kinase. The IMCD is the first polarized epithelium in which an ion channel has been shown to be directly regulated by a G protein. Thus, G proteins are important elements in regulating sodium absorption by the IMCD.