Karl L. Skorecki

Reduced cyclosporin accumulation in multidrug-resistant cells.

Cyclosporin accumulation was reduced by 50% or more in multidrug- resistant CHRC5 CHO cells with high levels of P-glycoprotein expression compared to drug sensitive AuxB1 CHO cells. This difference could be overcome by verapamil which is known to interact with P-glycoprotein and reverse multidrug resistance. The difference in cyclosporin accumulation between sensitive and resistant cells decreased with increasing cyclosporin concentrations suggesting that cyclosporine itself regulated its own accumulation through interaction with P-glycoprotein. Indeed, cyclosporin also reversed differences in vinblastine accumulation between resistant and sensitive cell lines. Since P-glycoprotein is highly expressed in the kidney which is also a target for cyclosporin toxicity, the effects of verapamil on cyclosporin accumulation were studied in two renal cell lines, rat mesangial cells and LLCPK1, cells. Verapamil increased cyclosporin accumulation by approximately 70%. These results suggest that cellular cyclosporine accumulation is regulated at least in part by its interaction with P-glycoprotein.

Cytosolic phospholipase A2 gene in human and rat: chromosomal localization and polymorphic markers

We report the chromosomal localization and a simple sequence repeat (SSR) in the cytosolic phospholipase A2 (cPLA2) gene in both human and rat. A (CA)18 repeat in the promoter of the rat gene was determined to exhibit length polymorphism when analyzed using the polymerase chain reaction (PCR) in 19 different inbred rat strains. Genotyping for this marker in 234 F2 progeny of a SHR x BN intercross mapped the gene to rat chromosome 13. Using a PCR strategy, a fragment of the promoter for the human gene was isolated, and a (CA)18 repeat was identified. Since this marker displayed a low heterozygosity index, we also identified a mononucleotide repeat in the promoter for cPLA2 that displayed a polymorphism information content value of 0.76. The human gene was mapped using fluorescence in situ hybridization (FISH) to chromosome 1q25. Of interest, the gene encoding the enzyme prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2), which acts on the arachidonic acid product of cPLA2, was previously localized to this same chromosomal region, raising the possibility of coordinate regulation. Identification of intragenic markers may facilitate studies of polymorphic variants of these genes as candidates for disorders in which perturbations of the eicosanoid cascade may play a role.

Body fluid homeostasis in congestive heart failure and cirrhosis with ascites

The urinary excretion of salt and water in man is regulated by a variety of renal and extrarenal mechanisms that respond to changes in dietary sodium intake as well as to alterations in the holding capacity of the vascular and interstitial compartments. Changes in extracellular fluid volume are detected by volume sensors located in the intrathoracic vascular bed, the kidney and other organs. These sensing mechanisms gauge the adequacy of intravascular volume relative to capacitance at various sites within the circulation. Congestive heart failure and cirrhosis with ascites are two disease states of man in which a hemodynamic disturbance within a given circulatory subcompartment is perceived by these sensing mechanisms and results in renal sodium retention. While the primary disturbance in both of these conditions originates outside the kidney, a variety of renal effector mechanisms respond to the perceived circulatory disturbance and result in enhanced tubule reabsorption of salt and water. These effector mechanisms involve physical adjustments in renal microvascular hemodynamics, tubule fluid composition and flow rate and transtubular ion gradients. These in turn are partially regulated by a variety of neural and humoral pathways including the renin-angiotensin-aldosterone axis, prostaglandins, and kinins.

Isolation of promoter for cytosolic phospholipase A2 (cPLA2)

Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid from membrane phospholipids and is believed to be the rate-limiting enzyme in the arachidonic acid pathway. We report herein the isolation of a 3 kb fragment of rodent genomic DNA containing part of the first intron, the first exon and 5-flanking sequence. The start site of transcription was mapped by 5-rapid amplification of cDNA ends and corroborated by ribonuclease protection assay. The gene has a TATAless promoter with no classical Sp1 binding sites or initiator element. A microsatellite series of CA repeats was noted in the 5-flanking region of both the rodent and human promoters. Deletion constructs have been analysed for luciferase activity and confirmed promoter activity.

Body fluid homeostasis in man: A contemporary overview

In the steady state, urinary excretion of sodium is closely matched to dietary salt intake. Given rigorous defense of extracellular fluid osmolality, it is the quantity of sodium in the extracellular fluid that determines the volume of this compartment. Changes in extracellular fluid volume are detected by volume sensors located in the intrathoracic vascular bed, kidney and other organs. These mechanoreceptors gauge the adequacy of intravascular volume, relative to capacitance, at various sites within the circulation. The perception of a change in the normal relationship between intravascular volume and circulatory capacity evokes a host of renal effector mechanisms that lead ultimately to physiologically appropriate changes in urinary sodium excretion. These effector mechanisms involve physical adjustments in the glomerular filtration rate, renal microvascular hemodynamics and peritubular capillary Starling forces, tubule fluid composition, flow rate and transtubular ion gradients. Neural and humoral pathways are also involved and, among the latter, angiotensin II, aldosterone, prostaglandins and kinins have been studied extensively. The continuous interaction between these sensor and effector mechanisms serves to ensure near-constancy of the extracellular fluid volume, a condition essential for optimal circulatory performance.

Muscle sympathetic nerve activity and renal responsiveness to atrial natriuretic factor during the development of hepatic ascites

PURPOSEnSodium retention in cirrhosis has been attributed to an imbalance between vasoconstrictive antinatriuretic forces such as the sympathetic nervous system and vasodilatory natriuretic agents such as atrial natriuretic factor (ANF). With the development of refractory ascites, cirrhotic patients become unresponsive to the natriuretic effect of ANF. Animal data suggest that the sympathetic nervous system plays a key role in mediating the refractoriness to ANF. We therefore studied the relationship between sympathetic nerve activity (SNA) and the natriuretic response to ANF in normal subjects and cirrhotic patients. We also attempted to localize the intrarenal site of refractoriness to ANF by lithium clearance.nnnPATIENTS AND METHODSnTwenty-six patients with biopsy-proven cirrhosis and seven age- and sex-matched normal volunteers were studied after a week of 20 mmol/day sodium intake and no diuretics. Muscle SNA was recorded from the peroneal nerve (microneurography) and correlated with responsiveness to a 2-hour ANF infusion. Lithium clearance was used as a marker of sodium reabsorption proximal to the intramedullary collecting duct, the main site of ANF action. Plasma norepinephrine, renin, and aldosterone levels were also determined. Patients were categorized into three groups: nine patients free of ascites (by ultrasonography), five ascitic patients who responded to a 2-hour ANF infusion (i.e., had a natriuretic response to ANF above 0.83 mmol/hour), and 12 ascitic patients who did not respond.nnnRESULTSnMuscle SNA was greatly increased in the ascitic nonresponder patients compared with the normal subjects (64 +/- 4 versus 27 +/- 7 bursts/minute, p less than 0.001), moderately increased in ascitic responders (47 +/- 6 bursts/minute, p less than 0.05), but not significantly increased in nonascitic patients with cirrhosis (34 +/- 5 bursts/minute). SNA was positively correlated with plasma norepinephrine levels (r = 0.69; p less than 0.005) and inversely correlated with peak sodium excretion during the ANF infusion (r = -0.63; p less than 0.001). Plasma renin activity and aldosterone were markedly elevated in ascitic nonresponders, and normal in ascitic responders and nonascitic patients. Lithium clearance was reduced in ascitic patients compared with nonascitic patients, did not change after the ANF infusion, and correlated inversely with SNA (r = -0.61; p less than 0.01).nnnCONCLUSIONnThese results support the concept that the sympathetic nervous system is a factor in renal sodium handling in cirrhosis, especially in the initiation of sodium retention and the development of refractory ascites. Refractoriness to ANF might be explained, at least in part, by increased neurally mediated sodium reabsorption proximal to the intramedullary collecting duct, the main site of ANF action.

Phase I-II study of vinblastine and oral cyclosporin A in metastatic renal cell carcinoma.

A phase I-II clinical trial was conducted to determine the maximum-tolerated dose (MTD) of oral cyclosporine (CsA) and vinblastine in patients with metastatic renal cell cancer (RCC) as well as to estimate the response rate. Sixteen patients received a 5 mg/kg oral loading dose of CsA followed by 3 days of CsA in 4 divided daily doses escalating from 10 mg/kg per day up to 17 mg/kg per day. Vinblastine (Vb1) was administered as an intravenous bolus on the morning of the 3rd day with dose escalation from 6 to 10 mg/m2. Cycles were repeated every 4 weeks until tumor progression. Forty-nine cycles of CsA with vinblastine were administered. The maximum tolerated dose of Vbl was 10 mg/m2. with neutropenia as the dose-limiting toxicity resulting in one death. CsA could not be escalated above 17 mg/kg per day because of nausea and vomiting. Other toxieities included constipation (100%). malaise (100%), temporary increase in pain (36%), and one seizure that may have been drug-related. There were no clinically significant changes in renal function or serum bilirubin. Mean peak whole-blood CsA level at the highest CsA dose level was 919 ng/ml (range: 414–1,827) with a trough prior to Vbl injection of 451 ng/ml (range: 128–1, 229). There were no tumor responses. The combination of oral CsA and Vbl is not nephrotoxic but is poorly tolerated. In most patients optimal blood levels of CsA for reversal of MDR cannot be reliably achieved, and vinblastine dose intensity must be compromised because of the significant toxicity of this regimen.

Inhibition of prostaglandin E2 synthesis by a blocker of epithelial chloride channels

Arginine-vasopressin (AVP) elicits a variety of responses in cultured rat mesangial cells, among them stimulation of prostaglandin biosynthesis and activation of Cl- channels. AVP produced an 11-fold increase over basal levels in prostaglandin E2 release from cultured mesangial cells. This response was completely inhibited by 25 microM indomethacin and 82 +/- 5% inhibited by 25 microM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) which is a potent blocker of epithelial Cl- channels. The IC50 for NPPB inhibition of prostaglandin E2 release was 8 microM. Indomethacin and NPPB at 25 microM also inhibited AVP-stimulated cellular accumulation of prostaglandin E2 by 98% and 79 +/- 7% respectively. The inhibitory effect of NPPB was not due to interference with the cellular response to AVP since at 50 microM it did not block AVP-stimulated release of arachidonate metabolites from cells metabolically labeled with [3H]-arachidonic acid. It is suggested that NPPB inhibition of prostaglandin E2 synthesis is at the cyclooxygenase level on the basis of its structural similarity to the fenamic acid type of cyclooxygenase inhibitors.

Vinblastine and erythromycin: an unrecognized serious drug interaction

Vinblastine and erythromycin are among the most commonly used chemotherapeutic and antimicrobial agents, respectively. No interaction between the two has ever been reported. Towards the end of a phase I study of vinblastine plus oral cyclosporin (to reverse multidrug resistance), three patients also received erythromycin to raise their cyclosporin levels. All developed severe toxicity consistent with a much higher vinblastine dose than was actually given. This apparent potentiation of vinblastine toxicity has not been previously described.

Interaction of atrial natriuretic peptide-stimulated guanylate cyclase and vasopressin-stimulated calcium signaling pathways in the glomerular mesangial cell

Receptors for atrial natriuretic peptide (ANP) have been demonstrated in renal mesangial cells as well as other cell types in the glomerulus. The biochemical basis for the effects of ANP on glomerular hemodynamics remains undefined. Using cultured rat glomerular mesangial cells, we demonstrated a concentration-dependent stimulation of cGMP production in intact cells, and of guanylate cyclase in membranes. Despite the presence of a guanylate cyclase response, ANP had no inhibitory effect on basal inositol trisphosphate production nor on basal cytosolic calcium. Arginine vasopressin stimulated IP3 production, caused a rise in cytosolic calcium as measured using the calcium-sensitive fluorescent probe Indo-1, and caused mesangial cell contraction. ANP caused a slight but significant enhancement of vasopressin-stimulated IP3 production, but had no effect on the cytosolic calcium response nor on the contractile response. 8-Bromo-cGMP likewise had no effect on the generation of the calcium signal. These results indicate that the effects of ANP on glomerular hemodynamics are not mediated by an alteration in the generation of the calcium signal in mesangial cells. In contrast, addition of calcium inhibited ANP stimulated guanylate cyclase activity.