Search PubMed⌕ Search

Biomedical subjects

W J Welch

Publications and source records attributed to W J Welch.

At least 19 recordsLinked to original sources
PubMed1992

Nitric oxide synthase in macula densa regulates glomerular capillary pressure.

Tubular-fluid reabsorption by specialized cells of the nephron at the junction of the ascending limb of the loop of Henle and the distal convoluted tubule, termed the macula densa, releases compounds causing vasoconstriction of the adjacent afferent arteriole. Activation of this tubuloglomerular feedback response reduces glomerular capillary pressure of the nephron and, hence, the glomerular filtration rate. The tubuloglomerular feedback response functions in a negative-feedback mode to relate glomerular capillary pressure to tubular-fluid delivery and reabsorption. This system has been implicated in renal autoregulation, renin release, and longterm body fluid and blood-pressure homeostasis. Here we report that arginine-derived nitric oxide, generated in the macula densa, is an additional intercellular signaling molecule that is released during tubular-fluid reabsorption and counters the vasoconstriction of the afferent arteriole. Antibody to rat cerebellar constitutive nitric oxide synthase stained rat macula densa cells specifically. Microperfusion of the macula densa segment of single nephrons with N omega-methyl-L-arginine (an inhibitor of nitric oxide synthase) or with pyocyanin (a lipid-soluble inhibitor of endothelium-derived relaxation factor) showed that generation of nitric oxide can vasodilate the afferent arteriole and increase glomerular capillary pressure; this effect was blocked by drugs that prevent tubular-fluid reabsorption. We conclude that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response. These findings imply a role for arginine-derived nitric oxide in body fluid-volume and blood-pressure homeostasis, in addition to its established roles in modulation of vascular tone by the endothelium and in neurotransmission.

Amino Acid Oxidoreductases↗
PubMed1992

Measurement of ventricular electrogram amplitude during intraoperative induction of ventricular tachyarrhythmias.

Adequate sensing of ventricular tachycardia (VT) and ventricular fibrillation (VF) is necessary for proper functioning of an implantable cardioverter defibrillator (ICD). Several ICDs currently undergoing investigation have programmable fixed gain sensitivity for tachycardia detection. If intracardiac electrogram amplitude decreases below the programmed sensitivity during VT or VF, detection of a ventricular arrhythmia may be delayed or missed. The mean amplitude of intracardiac electrograms (ICEGM) recorded with bipolar epicardial or transvenous sensing leads was measured in 63 patients during induced VT and VF recorded in the operating room at the time of ICD implantation. The mean amplitude of the ICEGM during 41 episodes of VF in 15 patients decreased from 14.9 +/- 0.9 mV during sinus rhythm to 8.8 +/- 0.7 mV at 1 second, 9.7 +/- 0.7 mV at 5 seconds, and 9.4 +/- 0.7 mV at 10 seconds (p < 0.0001 vs sinus rhythm ICEGM) with endocardial leads. The mean amplitude of the ICEGM recorded during 173 episodes of VF in 43 patients with epicardial leads decreased from 10.4 +/- 0.3 mV in sinus rhythm to 7.8 +/- 0.3 mV at 1 second, 8.3 +/- 0.3 mV at 5 seconds and 8 mV at 10 seconds (p <0.0001 vs sinus rhythm ICEGM). The mean amplitude of epicardial and transvenous ICEGMs recorded during 34 episodes of monomorphic VT decreased from 18.5 +/- 1.8 mV (epicardial) and 14.4 +/- 2.0 mV (transvenous) during sinus rhythm (p = 0.15, epicardial vs transvenous) to 16.0 +/- 1.7 mV (epicardial) and 13.7 +/- 1.9 mV (transvenous) at 10 seconds (< 10% of baseline amplitude).(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac Pacing, Artificial↗
PubMed1992

Response of human breast cancer cells to heat shock and chemotherapeutic drugs.

Previous studies have shown that certain chemotherapeutic drugs are less effective on tumor cells when cells have been previously exposed to hyperthermia. In the present study, we have evaluated whether specific modifications in heat shock protein (hsp) expression are associated with resistance to anticancer drugs. RNA levels for hsp90, hsp70, and hsp27 were studied by Northern and slot blots, while proteins were studied by two-dimensional gel electrophoresis, in MCF-7/BK and MDA-MB-231 breast cancer cells. The sensitivities of these cells to doxorubicin, colchicine, 5-fluorouracil, cisplatin, actinomycin D, and methotrexate were tested by clonogenic assays. These techniques were applied to both cell lines before (control) and after heat shock. The study revealed that elevated hsp70 and hsp27 levels were associated with doxorubicin resistance. In addition, the presence of phosphorylated hsp27 isoforms was also associated with doxorubicin resistance. The study showed that elevated hsps were not associated with multidrug resistance. Heat shock did not induce P170 glycoprotein mRNA overexpression or resistance to the other drugs tested. We also found that the level of doxorubicin protection conferred by the overexpression of hsp was lower than that obtained in cells expressing a multidrug resistance phenotype (MDA-A1R cells). In these cells, heat shock did not confer additional doxorubicin resistance and hsp27 phosphorylation was deficient. Our studies suggest that specific hsps are associated with doxorubicin resistance in certain human breast cancer cells and that this mechanism seems to be independent of the multidrug resistance system.

Breast Neoplasms↗
PubMed1992

Growth arrest of human B lymphocytes is accompanied by induction of the low molecular weight mammalian heat shock protein (Hsp28).

A large number of protein and molecular markers have been identified that delineate the early stages of human B cell activation and proliferation. In contrast, few if any molecules are transiently expressed precisely as activated B cells stop proliferating and undergo growth arrest. We demonstrate that the low molecular weight heat shock protein (hsp28) exhibits unique induction kinetics that specifically demarcates this interval. After mitogenic activation of unstimulated splenic B cells, hsp28 protein and phosphorylation transiently increase coinciding precisely with the peak of cellular proliferation and the onset of growth arrest. Although most neoplastic B cells constitutively express hsp28, three cell lines were identified that were hsp28-. No differences in phenotype or growth kinetics were detected between hsp28+ and hsp28- neoplastic B cells demonstrating that hsp28 expression is not essential for cell growth. However, when treated with phorbol ester or heat shock, these hsp28- cell lines synthesize hsp28 followed by the onset growth arrest. The consistency with which hsp28 induction transiently delineates the interval from peak proliferation to the onset of growth arrest suggests hsp28 itself is likely to be involved in regulating this process.

B-Lymphocytes↗
PubMed1992

Heat shock alters the composition of heteromeric steroid receptor complexes and enhances receptor activity in vivo.

Under normal cellular conditions, human progesterone receptors (PR), immune-isolated from cytosols of T47D breast cancer cells, associate with two heat shock proteins (hsps), hsp 90 and hsp 70. Receptors activated by hormone binding in vivo and extracted from nuclei with 0.5 M NaCl no longer associate with hsp 90 but retain association with hsp 70. We have examined the effect of heat shock treatment of cells on hsp-receptor interactions and on receptor function. Heat shock resulted in a partial reduction in cellular levels of PR, but receptors that remained were functional for both steroid and DNA binding activities. By steady-state [35S]methionine labeling prior to heat shock treatment, it was determined that heat shock did not affect the composition or maintenance of preexisting cytosolic PR.hsp 90.hsp 70 complexes. By contrast, immune isolation of PR complexes from cells pulse-labeled with [35S]methionine showed that heat shock altered the composition of newly synthesized hsps associated with PR. After heat shock, both the highly inducible form of hsp 70 (72K hsp) and a 100K hsp were bound to cytosol PR, and inducible 72K hsp remained bound with the nuclear-activated PR. Neither of these hsps were associated in detectable amounts with PR under normal cellular conditions. With respect to receptor function, heat shock treatment substantially enhanced the activity of PR in vivo as determined by measuring hormone-dependent PR-mediated transcription of a target reporter gene (MMTV-CAT) that was stably transfected into T47D cells. Heat shock treatment alone, in the absence of hormone, did not stimulate MMTV-CAT expression nor did it affect transcription from a control reporter gene, pSV2-CAT, suggesting that enhanced receptor activity was due to an effect on PR-mediated processes and not to a general effect on transcription. Induction of the heat shock response by a related chemical stress (sodium arsenite) also enhanced PR activity in vivo. Interestingly, sodium arsenite produced both a greater induction of hsp 90 and hsp 70 synthesis and a greater fold enhancement of PR-mediated gene transcription than did heat shock. This suggests that enhancement of PR activity is related not only to induction of hsp synthesis but also to the severity of the stress response. The present results provide an indication that in certain cells there may exist an interrelationship between the activation pathways by which cells respond to stress and to steroid hormones. Possible mechanisms responsible for heat shock effects on PR activity are discussed.

Arsenic↗
PubMed1992

Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events.

To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: 1) absence of progesterone, 2) elevated temperature (30 degrees C), 3) presence of ATP, and 4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP-regenerating system. ATP depletion of lysate by dialysis or by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors.

Adenosine Triphosphate↗
PubMed1992

Implantation of cardioverter defibrillators in the post-sternotomy patient.

In an attempt to minimize the hazards of redo sternotomy or thoracotomy in patients who have undergone previous cardiac procedures, a technique has been developed for cardioverter defibrillator implantation that involves dissection through a left subcostal incision and placement of extrapericardial defibrillation patches. This approach was used in 22 consecutive patients who required an implantable cardioverter defibrillator 4 to 156 months after previous median sternotomy. Defibrillation threshold energy was less than or equal to 20 J in every patient. Ninety-one percent of patients were extubated during the first 24 hours and were transferred out of the intensive care unit by the second postoperative day. One patient died of an acute myocardial infarction 3 days postoperatively (1/22, 4.5%). It was necessary to replace one lead for mechanical failure of an adapter, one patch required repositioning, and 1 patient needed drainage of a persistent pleural effusion (3/22, 13.6%). No further complications occurred during 3 to 27 months of follow-up. Advantages of the subcostal approach included prompt extubation, a single incision, and minimal morbidity. This approach is safe and effective, and is the method of choice for implantation of a cardioverter defibrillator in patients who have undergone prior sternotomy.

Adult↗
PubMed1992

Examining the function and regulation of hsp 70 in cells subjected to metabolic stress.

Members of the heat-shock protein (hsp) 70 family, distributed within various cellular compartments, have been implicated in facilitating protein maturation events. In particular, related hsp 70 family members appear to bind nascent polypeptides which are in the course of synthesis and/or translocation into organelles. We previously reported that in normal, unstressed cells, cytosolic hsp 70 (hsp 72/73) interacted transiently with nascent polypeptides. We suspect that such interactions function to prevent or slow down the folding of the nascent polypeptide chain. Once synthesis is complete, and now with all of the information for folding present, the newly synthesized protein appears to commence along its folding pathway, accompanied by the ATP-dependent release of hsp 72/73. Herein, we examined how these events occur in cells subjected to different types of metabolic stress. In cells exposed to either an amino acid analog or sodium arsenite, two potent inducers of the stress response, newly synthesized proteins bind to but are not released from hsp 70. Under these conditions of metabolic stress, we suspect that the newly synthesized proteins are unable to commence proper folding and consequently remain bound to their hsp 70 chaperone. In cells subjected to heat shock, a large number of both newly synthesized as well as mature proteins are rendered insoluble. Within this insoluble material are appreciable amounts of hsp 72/73. Finally, we show that in cells depleted of ATP, the release of hsp 70 from maturing proteins is inhibited. Thus, in cells experiencing metabolic stress, newly synthesized proteins unable to properly fold, as will as mature proteins which begin to unfold become stably bound to hsp 72/73. As a consequence and over time, the free or available levels of pre-existing hsp 72/73 are reduced. We propose that this reduction in the available levels of hsp 72/73 is the trigger by which the stress response is initiated.

Adenosine Triphosphate↗
PubMed1992

Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor.

Heat shock genes encode proteins (hsp's) that play important structural roles under normal circumstances and are essential to the cells' ability to survive environmental insults. Evidence is presented herein that transcriptional regulation of hsp gene expression is linked with the regulation of overall protein synthesis as well as with the accumulation of proteins denatured by stressful events. The factor that connects the three processes appears to be one of the hsp's, presumably a member(s) of the hsp70 family. Biochemical experiments demonstrate that complexes containing hsp70 and heat shock transcription factor, the specific regulator of hsp gene activity, are formed in the cells.

Base Sequence↗
PubMed1992

Immunocytochemical response of type A and type B intercalated cells to increased sodium chloride delivery.

Two populations of intercalated cells, type A and type B, are present in the rat cortical collecting duct (CCD). Type A cells are involved in proton secretion and contain an apical H(+)-adenosinetriphosphatase (ATPase) and a basolateral Cl(-)-HCO3- exchanger. Type B cells are believed to be involved in HCO3- secretion, which is mediated by a Cl(-)-HCO3- exchange process and is Cl- dependent. The aim of this study was to examine the morphological and immunocytochemical response of type B intercalated cells in the rat to increased delivery of Cl- to the CCD. This was accomplished by chronic infusion of a loop diuretic, bumetanide (30 mg.kg body wt-1.day-1), via an osmotic minipump, and simultaneous administration of 0.9% sodium chloride in the drinking water for 6 days. The kidneys were preserved by in vivo perfusion with a periodate-lysine-paraformaldehyde fixative and processed for horseradish peroxidase and protein A gold immunocytochemistry, using rabbit polyclonal antibodies against carbonic anhydrase II, proton ATPase, and band 3 protein. Chronic infusion of bumetanide in combination with a high salt intake was associated with significant changes in the intercalated cells. Type B cells were increased in size and exhibited numerous apical microvilli, increased basolateral membrane area, and marked cytoplasmic and basolateral labeling for H(+)-ATPase. In contrast, type A cells were small and had sparse apical microprojections. H(+)-ATPase immunolabeling was observed primarily over apical tubulovesicles, and there was decreased basolateral immunolabeling for band 3 protein and occasional labeling for band 3 in lysosome-like structures. These observations support the hypothesis that increased delivery of Cl- to the CCD is associated with stimulation of type B intercalated cells to secrete HCO3-. The observations in type A cells are consistent with the cells being in a resting or inactivated state.

Adenosine Triphosphatases↗
PubMed1992

Heat shock protein induction in rat hearts. A role for improved myocardial salvage after ischemia and reperfusion?

BACKGROUND: To test the hypothesis that the heat shock response is associated with improved myocardial salvage after myocardial ischemia and reperfusion, rats treated with prior whole-body hyperthermia and 24 hours of recovery (n = 26) or 20 minutes of ischemic pretreatment and 8 hours of recovery (n = 24) and control rats (n = 27, n = 24, for hyperthermic and ischemic pretreatment, respectively) were subjected to 35 minutes of left coronary artery (LCA) occlusion and 120 minutes of reperfusion. METHODS AND RESULTS: Although ventricular samples from rats subjected to either hyperthermia (n = 7) or ischemic pretreatment (n = 6) all showed induction of HSP72 (heat shock protein), Western blot analysis revealed significantly greater amounts of HSP72 in samples obtained from rats subjected to hyperthermia compared with those from rats subjected to ischemic pretreatment. Control rats (n = 7) showed no significant presence of myocardial HSP72. After 35 minutes of LCA occlusion and 2 hours of reperfusion, infarct size was significantly reduced in heat-shocked rats compared with controls (8.4 +/- 1.7%, n = 26 versus 15.5 +/- 1.9%, n = 27; p = 0.007; mean +/- SEM; infarct mass/left ventricular mass x 100). There were no significant differences in left ventricular (LV) systolic pressure, heart rate, LV dP/dt, or rate-pressure product between heat-shocked (n = 11) and control (n = 14) rats during the ischemic period. There were no differences in infarct size between ischemically pretreated and control rats subjected to 35 minutes of ischemia and reperfusion (9.7 +/- 2.1%, n = 23 versus 10.0 +/- 2.1, n = 24; p = NS). CONCLUSIONS: In this model of ischemia and reperfusion, prior heat shock was associated with significantly improved myocardial salvage after 35 minutes of LCA occlusion and reperfusion. This improved salvage was correlated with marked HSP72 induction and was independent of the hemodynamic determinants of myocardial oxygen supply and myocardial oxygen demand during the ischemic period. In contrast, mild HSP72 induction by ischemic pretreatment was not associated with improved myocardial salvage after myocardial ischemia and reperfusion. Thus, the absolute levels of HSP72 may be important in conferring protection from ischemic injury in this animal model.

Animals↗
PubMed1992

Potentiation of tubuloglomerular feedback in the rat by thromboxane mimetic. Role of macula densa.

UNLABELLED: Because endogenous thromboxane A2 (TXA2) potentiates the tubuloglomerular feedback response (TGF), we studied the mechanism of action of TXA2 by using a stable TXA2/prostaglandin (PG) H2 mimetic, U-46,619. Intravenous infusion of U-46,619 at 100 ng.kg-1.min-1 reduced the GFR and the single-nephron (SN)GFR measured from the distal tubule (macula densa function intact), whereas the SNGFR measured from the proximal tubule (macula densa function interrupted) was not changed consistently. 10-100-fold higher rates of infusion of U-46,619 were required to raise blood pressure or femoral vascular resistance. The regulation of glomerular capillary pressure (PGC) by TGF was assessed in anesthetized rats from changes in proximal stop flow pressure (PSF) and/or SNGFR during perfusion of the loop of Henle (LH) with artificial tubular fluid (ATF). Orthograde loop perfusion and retrograde perfusion of U-46,619 into the macula densa segment reduced PSF. Responses to luminal U-46,619 were blunted by a TXA2-PGH2 receptor antagonist. Orthograde loop perfusions with luminal U-46,619 increased net Cl absorption, whereas coperfusion with furosemide (10(-4) M) blunted the response to U-46,619 by 68%. These data indicated that the luminal U-46,619 might increase the signal for TGF activation by increasing Cl reabsorption in macula densa cells. However, since 80 +/- 4% of [3H]U-46,619 perfused via the LH was reabsorbed peritubular capillaries (PTC) were perfused with U-46,619 to test additional extra-luminal actions. PTC perfusion with U-46,619 again increased TGF by reducing PSF selectively only while macula densa function was intact during perfusion of the LH with ATF. CONCLUSIONS: (a) TGF is potentiated by U-46,619 given systematically, via the lumen of the LH by orthograde or retrograde perfusions or via the PTC; (b) at the lower doses tested, reduction of PGC and SNGFR by U-46,619 depends on tubular fluid delivery and reabsorption by the macula densa; (c) potentiation of TGF by U-46,619 entails preglomerular vasoconstriction which may be elicited in part by an increased signal due to increased net chloride reabsorption in the LH and presumably macula densa cells and by an increased sensitivity of the arteriole to macula densa-derived signals; (d) activation of TGF may contribute to the selective vasoconstriction of the renal vascular bed by low doses of U-46,619.

Animals↗
PubMed1991

Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding.

Circular dichroism and HPLC gel filtration were used to show that cytosolic hsp70 is thermally stable but undergoes a conformational transition (midpoint, 43 degrees C; 57 degrees C in the presence of ATP or ADP) leading to oligomerization. hsp70 binds to unfolded, but not to folded, proteins in a temperature-dependent manner; complex formation is significant only at physiologically relevant temperatures. hsp70 binds ADP more tightly than ATP to form a binary complex, which binds to the unfolded protein more rapidly than free hsp70. ADP also inhibits the ATP-induced dissociation of the hsp70-protein complex. A regulatory role for the hsp70-nucleotide binary complexes is proposed.

Adenosine Diphosphate↗
PubMed1991

Characterization and purification of the 94-kDa glucose-regulated protein.

Increased synthesis of so-called glucose-regulated proteins (grp) of 78 and 94 kDa occurs in mammalian cells exposed to a variety of agents, including 2-mercaptoethanol, tunicamycin, agents which perturb calcium homeostasis, and amino acid analogs. Herein we describe a number of properties of 94-kDa grp (grp 94) and present a method for its purification to homogeneity. The protein, within the endoplasmic reticulum (ER), is modified by the addition of high mannose-containing oligosaccharides. The predicted amino acid sequence of grp 94, as determined by others, has revealed the protein to contain a putative transmembrane domain near its amino terminus, but in addition, a potential endoplasmic reticulum retention sequence (KDEL) at its COOH terminus. Consequently, the question of whether grp 94 exists as a transmembrane or luminal protein of the ER remains controversial. Results using isolated microsomes subjected to either limited proteolysis or lactoperoxidase-mediated iodination were consistent with the idea that the grp is a transmembrane protein. On the other hand, using the method of sodium carbonate extraction, grp 94 exhibited properties of both a luminal and integral membrane protein. These results raise the question of whether there exist two different forms of grp 94 within the ER.

Animals↗
PubMed1991

Autoantibodies to human stress proteins. A survey of various rheumatic and other inflammatory diseases.

Unselected sera from patients with various rheumatic, inflammatory bowel, and autoimmune skin diseases (n = 268) were examined against human cell lysate by immunoblotting procedures, to determine the prevalence of autoantibodies to stress proteins (heat-shock proteins) hsp60 (homolog of Escherichia coli groEL and mycobacterial 65K antigens), hsp73, and hsp90. Using standard, sensitive and specific assay conditions, IgG and IgM autoantibodies to these stress proteins were not demonstrable, or were detected infrequently, in sera from control subjects (n = 36) and from patients with rheumatoid arthritis, Sjögren's syndrome, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, and systemic sclerosis. Autoantibodies to hsp60 were relatively more common (greater than or equal to 20% of sera) in patients with mixed connective tissue disease, polymyositis/dermatomyositis, psoriatic arthritis, inflammatory bowel disease, epidermolysis bullosa acquisita, and bullous pemphigoid. Anti-hsp73 autoantibodies were detected in 20% or more of the sera from patients with Lyme disease and ulcerative colitis. Taken together, these data extend the spectrum of autoimmune and inflammatory diseases in which humoral anti-stress protein autoreactivity develops. However, the paucity of humoral autoreactivity to stress proteins in patients with systemic lupus erythematosus and rheumatoid arthritis argues against a direct role of anti-stress protein autoantibodies in the pathogenesis of these disorders.

Autoantibodies↗