Regulation of Na(+)-K(+)-2Cl- cotransporter activity in rat skeletal muscle and intestinal epithelial cells.

In mammalian cells, Na(+)-K(+)-2Cl- cotransporter activity participates in regulation of ion and volume homeostasis. This makes NKCC indispensable for normal cell growth and proliferation. We recently reported the existence of two mechanisms that can regulate NKCC activity in mature skeletal muscle. In isosmotic conditions, signaling through ERK MAPK pathway is necessary, while inhibition of the cAMP-dependent protein kinase A (PKA) pathway stimulates NKCC activity during hyperosmotic challenge. Both pathways are involved in regulating cell proliferation in wide variety of cells of epithelial and non-epithelial origin, so we tested which pathway regulated NKCC activity in cultured cells. In cultured rat skeletal muscle (L6) and intestinal epithelial (IEC-6) cells, NKCC activity in the basal state comprised 30 to 50% of total potassium influx, assessed as bumetanide-sensitive 38Rb-uptake. This NKCC activity could not be abolished by inhibitors of ERK MAPK (PD98059 and U0126), PKC (GF109203X), or PI 3-K (wortmannin, LY294002). In L6 myoblasts and in IEC-6 cells, elevation of cAMP levels with isoproterenol or forskolin led to a 60% inhibition on NKCC activity. In contrast, incubation of IEC-6 cells with the PKA-inhibitor H-89 resulted in 50% increase of NKCC activity compared with the basal level. In conclusion, it appears that in cultured cells the cAMP--PKA pathway regulates NKCC activity. This resembles hyperosmotic regulation of NKCC activity.

Fludarabine and melphalan-based conditioning for patients with advanced hematological malignancies relapsing after a previous hematopoietic stem cell transplant.

Severe regimen-related toxicity often complicates second transplant procedures performed in patients with hematological malignancies that have relapsed after an initial hematopoietic stem cell (HSC) transplant. Therefore, we studied the safety and efficacy of a reduced-intensity fludarabine and melphalan based conditioning regimen in 11 patients who had relapsed following an autologous (n = 7) or allogeneic (n = 4) HSC transplant. All patients received allogeneic peripheral blood HSC from either an HLA-identical (n = 7) or an HLA-mismatched (n = 4) relative. Diagnoses included AML (n = 9), ALL (n = 1), or Hodgkin's disease (n = 1). Only one patient was in complete remission at the time of second transplant. The median interval between first transplant and relapse was 163 days (range 58-1885). Recipients of HLA-mismatched transplants received antithymocyte globulin in addition to fludarabine and melphalan as part of the conditioning regimen. All 11 patients received acute GVHD prophylaxis consisting of tacrolimus and methotrexate. Ten of 11 patients achieved hematopoietic engraftment with a median time to absolute neutrophil count >0.5 x 10(9)/l and to platelet count of >20 x 10(9)/l of 14 and 19 days, respectively. All engrafting patients achieved 100% donor chimerism on initial analysis, except for one with persistent leukemia at day +19. Two patients experienced grade 3 regimen-related toxicity, manifesting as acute renal failure. Acute GVHD grades 2-4 occurred in two recipients and chronic GVHD in four. The 100-day mortality from all causes was 36%. Ten of 11 patients (91%) died a median of 140 days (range 9-996) after the second transplant. The causes of death included relapse (n = 5), sepsis (n = 4), and idiopathic pneumonia syndrome (n = 1). One patient with AML survives in remission at 880 days post-transplant. We conclude that fludarabine- and melphalan-based conditioning promotes full donor chimerism, even following HLA-mismatched transplants. However, the regimen may be more beneficial when applied to patients undergoing allogeneic HSC transplantation earlier in their disease course.

Insulin-independent, MAPK-dependent stimulation of NKCC activity in skeletal muscle.

Na(+)-K(+)-Cl(-) cotransporter (NKCC) activity in quiescent skeletal muscle is modest. However, ex vivo stimulation of muscle for as little as 18 contractions (1 min, 0.3 Hz) dramatically increased the activity of the cotransporter, measured as the bumetanide-sensitive (86)Rb influx, in both soleus and plantaris muscles. This activation of cotransporter activity remained relatively constant for up to 10-Hz stimulation for 1 min, falling off at higher frequencies (30-Hz stimulation for 1 min). Similarly, stimulation of skeletal muscle with adrenergic receptor agonists phenylephrine, isoproterenol, or epinephrine produced a dramatic stimulation of NKCC activity. It did not appear that stimulation of NKCC activity was a reflection of increased Na(+)-K(+)-ATPase activity because insulin treatment did not stimulate NKCC activity, despite insulin's well-known stimulation of Na(+)-K(+)-ATPase activity. Stimulation of NKCC activity could be blocked by pretreatment with inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) activity, indicating that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) MAPKs may be required. These data indicate a regulated NKCC activity in skeletal muscle that may provide a significant pathway for potassium transport into skeletal muscle fibers.

Osteopontin expression in spontaneously developed neointima in fowl (Gallus gallus).

Fowl show spontaneous elevation of blood pressure and neointimal plaque formation in the abdominal aorta at young ages. A similar neointima can be induced by a balloon-catheter-induced endothelium injury to the fowl aorta. Both spontaneously developed and injury-induced vascular lesions exhibit subendothelial hyperplasia consisting of neointimal cells with a synthetic phenotype and abundant extracellular matrix. The role of the extracellular matrix in the formation of neointima is not known. In this study, we investigated whether osteopontin, an adhesive glycoprotein present in the extracellular matrix, is expressed in aortic smooth muscle tissue of the fowl abdominal aorta, in spontaneously developed neointimal plaques and in the aortic smooth muscle underlying neointimal plaques. Crude protein extracted from isolated aortic smooth muscle tissues and neointimal plaques was fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with rabbit anti-fowl osteopontin (provided by Dr L. C. Gerstenfeld, Boston University) or anti-&agr; smooth muscle actin antibodies. The anti-fowl osteopontin antibody predominantly recognized a 66-70 kDa protein band in neointimal plaques that co-migrated with the osteopontin phosphoprotein from chick bone. In contrast, intact aortic smooth muscle and the smooth muscle underlying neointimal plaques equally expressed three proteins (66-70 kDa, approximately 50 kDa and approximately 43 kDa) recognized by the anti-osteopontin antibody. Anti-&agr; smooth muscle actin antibody recognized a 43 kDa protein band, and the expression of &agr; smooth muscle actin was higher in aortic smooth muscle than in neointimal plaques. Osteopontin mRNA expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) of total RNA from vascular tissues with specific primers constructed on the basis of the reported fowl osteopontin nucleotide sequence. The PCR products from intact aortic smooth muscle and neointimal plaques correspond to the product from recombinant plasmid cDNA (a gift from Dr L. C. Gerstenfeld) transcribed in vitro. These results suggest that osteopontin is synthesized in intact aortic smooth muscle and neointimal plaques in fowl and that unmetabolized approximately 66 kDa osteopontin protein is a predominant form in the neointima, indicating that osteopontin protein may be actively synthesized in the neointima.

Molecular and functional evidence for Na(+)-K(+)-2Cl(-) cotransporter expression in rat skeletal muscle.

Doubt has been raised about the expression of a functional Na(+)-K(+)-2Cl(-) cotransporter in rat skeletal muscle. In this study we present molecular and functional evidence for expression of a protein having the characteristics of a cotransporter. RT-PCR of RNA isolated from rat soleus muscle with primers to a conserved putative membrane-spanning domain resulted in a single product of predicted size. Sequencing of the product showed that it bears >90% homology with known rodent NKCC1 (BSC2) cotransporters. RNase protection assay of RNA isolated from the rat soleus muscle also identified this sequence. Immunologic detection of the cotransporter with two different antibodies indicated the presence of cotransporter protein, perhaps more than one, in blots of total muscle protein. Immunohistochemical detection by confocal microscopy localized the majority of expression of the protein to the muscle fibers. Functional studies of cotransport activity also indicate the appropriate sensitivity to inhibitors and ion dependence. Taken together, these data support the presence and function of Na(+)-K(+)-2Cl(-) cotransporter activity in the soleus muscle of the rat.

Unique 5'-end of a Na(+)-K(+)-2Cl- cotransporter-like mRNA expressed in rat skeletal muscle.

Functional evidence presented by others indicates that rat slow-twitch skeletal muscle lacks typical Na(+)-K(+)-2Cl- cotransporter activity, as determined by loop diuretic-sensitive potassium transport. This report presents a unique 5' mRNA sequence of a Na(+)-K(+)-2Cl- cotransporter-like molecule expressed in the rat soleus muscle and the deduced N-terminus of the protein. In addition to its unique 5' mRNA sequence, the coding region of the N-terminus is quite short compared with other known Na(+)-K(+)-2Cl- cotransporters. Nonetheless, the mRNA possesses conserved cotransporter-like membrane spanning domains, though one domains corresponding to a reported exon is divergent. Therefore, it appears that skeletal muscle does express a Na(+)-K(+)-2Cl- cotransporter-like mRNA that may code for a protein with atypical Na(+)-K(+)-2Cl- cotransporter properties.

Impact of genetic polymorphisms of the beta2-adrenergic receptor on albuterol bronchodilator pharmacodynamics.

OBJECTIVE: To determine whether genetic polymorphisms of the beta2-adrenergic receptor gene affect the relationship between albuterol (INN, salbutamol) plasma concentrations and the forced expiratory volume in 1 second (FEV1) in subjects with moderate asthma. METHODS: Sixteen clinically stable patients with moderate asthma who participated in a pharmacokinetic-pharmacodynamic study of albuterol volunteered to provide a blood sample for determination of beta2-adrenergic receptor genotype. FEV1 and plasma concentrations of albuterol were determined at various times after administration of an oral solution that contained 8 mg albuterol. Patients withheld inhaled beta2-agonist and corticosteroid therapy 12 and 24 hours, respectively, before the study. beta2-Adrenergic receptor genotype was determined by polymerase chain reaction with allele-specific oligonucleotide hybridization. RESULTS: Albuterol-evoked FEV1 was higher and the response was more rapid in Arg16 homozygotes compared with the cohort of carriers of the Gly16 variant: Maximal percentage increase in FEV1 (%deltaFEV1), 18% versus 4.9% (P < .03); area under FEV1 albuterol concentration curve, 194%.mL/ng versus 30%.mL/ng (P < .05); initial slope (dE/dC), 1.43%.mL/ng versus 0.55%.mL/ng (P < .003). CONCLUSIONS: The beta2-adrenergic receptor gene polymorphism is a major determinant of bronchodilator response to albuterol. Future pharmacodynamic studies of beta2-agonists should include determination of 02-adrenergic receptor genotype.

Beating affects the posttranscriptional regulation of alpha-myosin mRNA in cardiac cultures.

Contractile arrest of cardiac myocytes results in increased abundance of alpha-myosin heavy chain (MHC) mRNA but decreased alpha-MHC protein content. Our aim is to determine the posttranscriptional mechanisms regulating alpha-MHC mRNA-protein uncoupling in cultured neonatal rat hearts during altered contractile activity. Spontaneously contracting myocytes were arrested by the use of verapamil (10 mumol/l; a Ca(2+)-channel blocker) or by 2,3-butanedione monoxime (5 mmol/l; a cross-bridge inhibitor). Inhibition of transcription with amanitin (0.5 mumol/l) decreased the alpha-MHC mRNA in normally beating myocytes to a minimal baseline. However, the alpha-MHC mRNA did not fall this low in amanitin-treated nonbeating myocytes. Concurrently, the alpha-MHC mRNA shifted toward a heavier polysome complex when beating was blocked. Together, these data suggest that contractile arrest regulates alpha-MHC mRNA abundance posttranscriptionally by stabilizing the message at the elongation phase of translation. These posttranscriptional regulatory steps are dependent on beating itself and are independent of Ca2+ entry.

Fractal analysis of cytoskeleton rearrangement in cardiac muscle during head-down tilt.

Head-down tilt by tail suspension of the rat produces a volume, but not pressure, load on the heart. One response of the heart is cytoskeleton rearrangement, a phenomenon commonly referred to as disruption. In these experiments, we used fractal analysis as a means to measure complexity of the microtubule structures at 8 and 18 h after imposition of head-down tilt. Microtubules in whole tissue cardiac myocytes were stained with fluorescein colchicine and were visualized by confocal microscopy. The fractal dimensions (D) of the structures were calculated by the dilation method, which involves successively dilating the outline perimeter of the microtubule structures and measuring the area enclosed. The head-down tilt resulted in a progressive decrease in D (decreased complexity) when measured at small dilations of the perimeter, but the maximum D (maximum complexity) of the microtubule structures did not change with treatment. Analysis of the fold change in complexity as a function of the dilation indicates an almost twofold decrease in microtubule complexity at small kernel dilations. This decrease in complexity is associated with a more Gaussian distribution of microtubule diameters, indicating a less structured microtubule cytoskeleton. We interpret these data as a microtubule rearrangement, rather than erosion, because total tubulin flourescence was not different between groups. This conclusion is supported by F-actin fluorescence data indicating a dispersed structure without loss of actin.

Decreased Ca2+ sensitivity of isometric tension in skinned cardiac myocytes from tail-suspended rats.

Tail suspension in rats causes a cephalic shift in blood, resulting in a volume load on the heart similar to that observed during microgravity spaceflight or mild heart failure. The present study determined the influence of increased cardiac hemodynamic load on myofilament isometric tension as a function of Ca2+ concentration in skinned cardiac myocytes of control and 7-day head-down tilt Sprague-Dawley rats. Isometric force of single skinned myocytes was measured by attaching cells with adhesive to a force transducer and piezoelectric translator. A significant decrease in the Ca2+ sensitivity of tension was observed in cardiac myocytes from suspended rats [pCa of half-maximal tension (pCa50) of 5.83 +/- 0.03] compared with control rats (pCa50 of 5.94 +/- 0.03). Maximum tension generation and slope of the tension-pCa relationship were unaffected by head-down tilt. Electrophoretic analysis of myofilament proteins indicates differences in expression of proteins in the 50-60 and 100-120 kDa ranges; immunoblot analysis of tubulin (50 kDa) expression indicates no change in the ratio of beta-tubulin to light chain 1 or tropomyosin. Decreased force-producing ability at a given submaximum Ca2+ concentration in cardiac myocytes from suspended rats suggests a decrease in contractility possibly due to changes in cardiac myofilament protein expression following chronic elevated volume load on the heart.

Head-down tilt increases rat cardiac muscle eIF-2 alpha phosphorylation.

We previously demonstrated that head-down tilt in rats decreases heart polypeptide initiation rate and proposed a mechanism whereby redistribution of the chaperone heat-shock cognate/heat-shock protein-70 (HSC/HSP-70) facilitates the phosphorylation of eukaryotic initiation factor-2 alpha (eIF-2 alpha). In this study, two-dimensional gel electrophoretic analysis of eIF-2 alpha showed no phosphorylation in control hearts. At 8 h of head-down tilt, there was a 45% increase in total eIF-2 alpha, and 79% was phosphorylated. At 18 h, eIF-2 alpha increased to 142% of control, of which 4% was phosphorylated. This is consistent with the previous study where, at 8 h, there was a 78% increase in polysomal HSC/HSP-70 and a shift in the polysome center-of-mass to lighter polysomes (indicating decreased initiation). After 18 h of suspension, polysomal HSC/HSP-70 levels were 24% relative to control, and the center-of-mass returned toward control. We conclude that the decrease in polypeptide initiation during head-down tilt is mediated by HSC/HSP-70 via phosphorylation of eIF-2 alpha.

Decreased polysomal HSP-70 may slow polypeptide elongation during skeletal muscle atrophy.

Slowed elongation rate is the apparent cause of the rapid decrease in rat soleus muscle protein synthesis rate during non-weight bearing. We found that elongation factor 2 was not phosphorylated and thus could not explain the slowed elongation rate. However, we observed a 44 +/- 19 and 28 +/- 14% decrease in the chaperone protein 70-kDa heat-shock cognate/heat shock protein (HSC/HSP-70) associated with the polysomes after 12 and 18 h of non-weight bearing, respectively. Size-fractionated polysomes had less HSC/HSP-70 associated with the larger polysomes in 18-h non-weight-bearing soleus muscle. ATP concentration increased in the non-weight-bearing muscle, so, because ATP enhances HSC/HSP-70 dissociation, we tested the potential role of ATP. Digitonin-permeabilized myoblasts treated with increasing concentrations of ATP showed both a decreased association of HSC/HSP-70 with the polysomes and a shift toward heavier polysomes; these responses were blocked by adenosine 5'-O-(3-thiotriphosphate). These data are consistent with the role of HSC/HSP-70 as a chaperone of nascent protein. The absence of HSC/HSP-70 may slow ribosome translocation, thus slowing elongation rate.

Decrease in heart peptide initiation during head-down tilt may be modulated by HSP-70.

This study examines the mechanism of the rapid decrease in cardiac muscle protein synthesis during rodent hindlimb non-weight bearing. Polysomes isolated from rat hearts 8 h after suspension show less RNA in the polysome pool and a shift in polysome size toward fewer ribosomes per mRNA; 18 h after suspension, the size shift persists, but the amount of RNA in the polysome pool returns to control values. These data are consistent with a decrease in the rate of initiation of protein synthesis. At both 8 and 12 h of suspension, the cardiac polysomes show a 78 and 93% increase association with the nascent polypeptide chaperone protein 70-kDa heat-shock cognate/heat-shock protein (HSC/HSP-70), respectively, that persists after 7 days of non-weight bearing. Because the dissociation of HSC/HSP-70 from unfolded protein can be modulated by ATP, we measured the adenosine nucleotide pools and found a 53% decrease in ATP levels after 18 h of suspension. We propose a mechanism in which a shift of HSC/HSP-70 to the nascent polypeptide indirectly inhibits protein synthesis initiation.

Soleus muscle nascent polypeptide chain elongation slows protein synthesis rate during non-weight-bearing activity.

Protein synthesis rate of the soleus muscle decreases rapidly during non-weight-bearing activity. We isolated polysomes from 18-h non-weight-bearing soleus muscle to investigate the mechanism of this phenomenon. The distribution of polysomal alpha-actin mRNA and 18S rRNA on sucrose density gradients shows that polysomes shift to larger sizes (more ribosomes per mRNA) during non-weight-bearing activity. Furthermore, RNA is mobilized into the polysome pool of the non-weight-bearing soleus muscle; these data indicate that initiation of protein synthesis is not rate limiting. We explain these results as the slowing of nascent polypeptide chain elongation, such that there is a "traffic jam" of ribosomes on the mRNAs, increasing the number of ribosomes per mRNA while, at the same time, decreasing protein synthesis rate. In support of this hypothesis, myoblasts treated with a low dose of cycloheximide (a specific elongation inhibitor) show a similar shift in polysome size. A numerical model of protein synthesis further shows that elongation is more effective than initiation and termination in affecting protein synthesis and polysome size. We conclude that the non-weight-bearing-induced decrease in postural muscle protein synthesis rate is initially caused by slowing of nascent polypeptide chain elongation.

An easily synthesized, photolyzable luciferase substrate for in vivo luciferase activity measurement.

Many reporter gene assays require killing the cell by fixation or lysis. For assays in living cells, the substrate delivery is inefficient and cannot be supplied in situ in a bolus, which makes assays highly variable. We report a simple synthesis of a luciferin ester that is both photolyzable and cleaved by endogenous esterases such that luciferase activity in living cells is easily monitored. Although the photolyzed substrate can be delivered in bolus, the rapid equilibration of the luciferin ester in the cell and the continuous delivery by the endogenous esterases allow stable, long-term measurements of luciferase activity.

Altered actin and myosin expression in muscle during exposure to microgravity.

The mechanism for cardiovascular deconditioning and skeletal muscle atrophy during microgravity is not known. The purpose of the present study was to determine whether a decrease in contractile protein gene expression in the muscle of rats occurred after 14 days of microgravity. No differences existed in the profile of myosin protein isoforms or beta-myosin heavy chain mRNA in hearts between the flight and synchronous control groups. On the other hand, differences in the expression of beta-myosin heavy chain mRNA relative to the 18S and 28S rRNA in the heart between flight and synchronous control groups were noted with a covariance mapping analysis. Both the vastus intermedius and lateral gastrocnemius muscles exhibited significant (P less than 0.05) decreases in skeletal alpha-actin mRNA per unit of extractable RNA in the flight group compared with the synchronous control group. However, no significant difference for skeletal alpha-actin mRNA occurred in the triceps brachii muscle between these groups. Cytochrome c mRNA per unit of extractable RNA decreased (P less than 0.05) only in the vastus intermedius but not in the lateral gastrocnemius or triceps brachii muscles. In summary, changes in the pretranslational regulation of contractile protein gene expression occur in both heart and skeletal muscle after 14 days of microgravity.

New nonpeptide angiotensin II receptor antagonists. 1. Synthesis, biological properties, and structure-activity relationships of 2-alkyl benzimidazole derivatives.

On the basis of an extension of the literature lead 1, a series of benzimidazoles have been synthesized and shown to be angiotensin II (AII) receptor antagonists. The structure-activity relationships of these new antagonists have been explored and the key binding interactions defined. Molecular mechanics calculations were carried out on analogues of imidazole AII antagonists and conformationally restricted analogues were synthesized. The benzimidazole antagonists displaced AII in binding studies in vitro with IC50 values in the range 10(-5)-10(-7) M and antagonized the hypertensive effects of AII in vivo (rats) following intravenous administration with ED50 values in the range of 5-20 mg/kg.

Intermittent acceleration as a countermeasure to soleus muscle atrophy.

The centrifuge proposed for the Space Station will most likely be used, in part, for countermeasure studies. At present, there is a paucity of information concerning the duration and frequency of acceleration necessary to counteract the atrophy process associated with microgravity. The present study was designed to investigate intermittent acceleration during non-weight bearing of the soleus muscle and its resultant effects on muscular atrophy. Each day rats were removed from hindlimbs suspension and accelerated to 1.2 g for four 15-min periods evenly spaced over a 12-h interval. The soleus muscle experienced non-weight bearing the remaining 23 h each day. This paradigm, when repeated for 7 days, did not completely maintain the mass of soleus muscle, which was 84% of control. Interestingly, the identical protocol utilizing ground support in lieu of acceleration successfully maintained the soleus muscle mass. The failure of the centrifugation protocol to adequately maintain soleus muscle mass might be due to an undefined stress placed on the animals inherent in centrifugation itself. This stress may also explain the transient decline in food intake of the intermittent acceleration group on the 2nd and 3rd days of treatment. Also, these data support the concept that the frequency of exposure, as opposed to the duration of exposure, to weight bearing during hindlimb unweighting seems to be the more important determinant of maintaining postural muscle mass.