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Biomedical subjects

R M Hays

Publications and source records attributed to R M Hays.

At least 55 records · Page 3Linked to original sources
PubMed1989

Atrial natriuretic peptide and nitroprusside cause relaxation of cultured rat mesangial cells.

These studies evaluate the effects of atrial natriuretic peptide (ANP) and nitroprusside (NP) on cultured mesangial cells (MC) grown on a flexible silicone rubber surface. The basal tone of the MC produced wrinkles on the silicone rubber surface. A decrease in number or magnitude of wrinkles was considered to represent cell relaxation, whereas an increase represented cell contraction. ANP (10(-9) M) produced a relaxation in greater than 60% of the cells by 10 min. The percentage of cells showing a decrease of wrinkles was significantly higher (P less than 0.05 at 5 min and P less than 0.001 at 10 min) during the ANP-treated period than during the control period. NP (10(-5) M) caused a decrease of wrinkles in greater than 80% of cells (P less than 0.02 at 5 min and P less than 0.01 at 10 min) compared with a 5% decrease in the control period. Dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP; 10(-4) M) also produced a decrease of wrinkles in 81% of the cells (P less than 0.02) compared with a 9% decrease in the control period. MC treated with ANP, NP, or DBcGMP and then labeled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-phallacidin did not show obvious alteration of morphology of actin filaments compared with untreated cells. ANP could inhibit as well as partially reverse the agonist (angiotensin II)-induced contractile response. ANP (10(-10)-10(-8) M) as well as NP (10(-5) M) increased intracellular cGMP content of MC (P less than 0.005) compared with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amanitins↗
PubMed1989

Active urea transport in toad skin is coupled to H+ gradients.

Active transport of urea exists inwardly through the skin of the toad Bufo viridis and is enhanced by adapting the toads to hypertonic saline solutions. In this paper, we report our studies on the energy coupling of this active transport system. We have shown previously that this system is independent of sodium transport. We thus studied the possibility of coupling to proton transport by studying the following: 1) the effect of acidifying the epithelium by means of CO2 and protonophores, 2) the effect of alkalinizing the epithelium with the anion-exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and by removal of chloride from the serosal medium, 3) the effect of inhibiting the proton-translocating ATPase of the skin, 4) dependence on mucosal pH. We found that 1) acidification of the epithelium by means of 5% CO2 and the protonophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nigericin inhibited urea influx; 2) alkalinization of the epithelium by means of the disulfonic stilbene DIDS and by removal of serosal chloride increased urea influx; 3) inhibition of the proton-translocating ATPase of the skin by means of 1,3-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide (NEM), and sodium orthovanadate inhibited urea transport; 4) urea influx was unaffected by alkalinizing the external medium to 8.5 or by acidifying to 6.5; it was significantly suppressed by a mucosal pH of 5.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Evidence that monensin inhibits vasopressin-stimulated water flow at an early step in the receptor-adenylate cyclase sequence.

Monensin, a highly selective sodium ionophore, inhibits vasopressin-stimulated water flow in toad urinary bladder pretreated with naproxen, an inhibitor of prostaglandin synthesis. Inhibition is partially dependent on the presence of sodium in the serosal medium, but not on serosal calcium. We have found that monensin does not inhibit water flow generated by forskolin, cyclic AMP, or isobutyl methyl xanthine (MIX); indeed, an enhancement of water flow was seen following cAMP and MIX, as well as following 0.2 microM forskolin. Our findings suggest that monensin uncouples the vasopressin-receptor-G protein-adenylate cyclase sequence at some early step, by a mechanism that remains unknown, but that may directly or indirectly involve intracellular sodium.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Role of vesicular transport in ADH-stimulated aggregate delivery.

It has been assumed from studies in toad bladder that antidiuretic hormone (ADH)-stimulated particle delivery to the luminal membrane is mediated by particle-carrying tubular structures (aggrephores). We report studies in frog and toad urinary bladder showing that vesicles, rather than aggrephores, appear to play the major role in particle delivery in the frog and that vesicle and aggrephore delivery proceed in parallel in the toad. Our principal evidence for this view is that in the frog, transmission electron microscopy shows virtually no fused aggrephores. Supporting evidence includes the following. 1) Freeze-fracture studies show that the diameters of fusion events delivering particles can be quite small, indicating that they are formed by fused vesicles rather than fused aggrephores. 2) A significant population of small fusion events is also seen in the toad, along with larger fusion events related to both aggrephores and large vesicles. 3) Surface aggregate areas in both species are small, consistent with vesicular delivery. 4) Freeze-fracture replicas indicate delivery from shallow pits. We propose a system of transport of particles in which aggrephores act largely as intermediate storage organelles in the frog and as storage and fusion organelles in the toad.

Animals↗
PubMed1988

Active urea transport by the skin of Bufo viridis: amiloride- and phloretin-sensitive transport sites.

Urea is actively transported inwardly (Ji) across the skin of the green toad Bufo viridis. Ji is markedly enhanced in toads adapted to hypertonic saline. We studied urea transport across the skin of Bufo viridis under a variety of experimental conditions, including treatment with amiloride and phloretin, agents that inhibit urea permeability in the bladder of Bufo marinus. Amiloride (10(-4) M) significantly inhibited Ji in both adapted and unadapted animals and was unaffected by removal of sodium from the external medium. Phloretin (10(-4) M) significantly inhibited Ji in adapted animals by 23-46%; there was also a reduction in Ji in unadapted toads at 10(-4) and 5 x 10(-4) M phloretin. A dose-response study revealed that the concentration of phloretin causing half-maximal inhibition (K1/2) was 5 x 10(-4) M for adapted animals. Ji was unaffected by the substitution of sucrose for Ringer solution or by ouabain. We conclude 1) the process of adaptation appears to involve an increase in the number of amiloride- and phloretin-inhibitable urea transport sites in the skin, with a possible increase in the affinity of the sites for phloretin; 2) the adapted skin resembles the Bufo marinus urinary bladder with respect to amiloride and phloretin-inhibitable sites; 3) we confirm earlier observations that Ji is independent of sodium transport.

Amiloride↗
PubMed1988

Actin filament morphology in living and nonliving cultured mesangial cells: formation and dissolution.

Mesangial cells are contractile and are believed to play a role in the regulation of glomerular filtration. Actin filaments constitute an integral part of the cytoskeleton of these cells. To visualize actin filaments in both living and fixed acetone-extracted (nonliving) mesangial cells, we have used the fluorophore nitrobenzoxadiazole-phallacidin. Under the fluorescence microscope, mesangial cells displayed continuous staining. Distinct linear structures running from one end to the other were actin filaments or stress fibers (SF). SF were rather feathery and of slightly curvilinear appearance in living versus fixed, acetone-extracted cells. Formation of SF started a 4 h, and the majority of the cells had well developed SF within 24 h. To find out whether adhesion to a substrate was necessary to SF formation, we plated mesangial cells on variable thickness of poly-HEMA(2-hydroxyethyl methacrylate). This plastic surface at a high concentration (10(-1) M) produces poor adherence of mesangial cells. At 10(-1) M of poly-HEMA, the cells were rounded even after 48 h and did not develop any SF. In contrast, at a lower concentration (10(-4) M), mesangial cells were well spread, and SF were readily observed. These data indicated that spreading as well as formation of SF were directly related to the adhesion properties of the substrate. To evaluate the role of SF in contraction and relaxation of mesangial cells, we have first treated these cells with angiotensin II (5 x 10(-7) M) or dibutyryl cyclic adenosine monophosphate (5 x 10(-4) M) and then labeled them with nitrobenzoxadiazole phallacidin. Dibutyryl cyclic adenosine monophosphate caused dissolution of SF.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1988

Evidence for immunoglobulin Fc receptor-mediated prostaglandin2 and platelet-activating factor formation by cultured rat mesangial cells.

The possibility of Fc-dependent uptake of IgG immune complexes was examined in subcultured rat mesangial cells free of monocytes. 195Au-labeled colloidal gold particles were coated either with BSA only or with BSA followed by rabbit anti-BSA-IgG or the F(ab')2 fragment of the IgG. Mesangial cells preferentially took up 195Au particles covered with BSA-anti-BSA-IgG over those covered with BSA or the F(ab')2 fragment. This uptake was a time-dependent and saturable process inhibitable by sodium azide or cytochalasin B. Using phase-contrast microscopy in the light reflectance mode, it was established that essentially all mesangial cells took up IgG-coated gold particles. By electron microscopy the process was shown to consist of vesicular uptake with delivery to endosomes. Mesangial binding-uptake of the IgG-covered particles was associated with stimulation of PGE2 synthesis and production of platelet-activating factor, a lipid mediator of inflammation. To characterize the potential Fc receptor for IgG we used the rosetting technique with sheep red blood cells coated with IgG subclass-specific mouse monoclonal antibodies. 50% of mesangial cells exhibited rosetting with red cells coated with mouse IgG2a, whereas negligible rosetting was observed with IgG2b or IgG1. Competition experiments confirmed the specificity of IgG2a binding. We conclude that cultured rat mesangial cells exhibit specific receptors for IgG and that occupancy of Fc receptors results in endocytosis and is associated with generation of PGE2 and platelet-activating factor. These observations may be of significance for immune-mediated glomerular diseases.

Animals↗
PubMed1987

Endocytosis by cultured mesangial cells and associated changes in prostaglandin E2 synthesis.

The mechanism of macromolecule uptake by cultured mesangial cells was studied by use of transmission electron microscopy. In parallel, we investigated the effect of macromolecular uptake on prostaglandin E2 (PGE2) formation. Cultured rat mesangial cells were studied in their third passage. As model molecules, we used colloidal gold particles (10 nm diameter) coated either with polyethylene glycol (PEG) or fresh serum (SCG). Mesangial cells were incubated from 1 to 60 min and up to 12 h with either PEG or SCG particles. Endocytosis of SCG significantly exceeded that of PEG particles. The mechanism involved binding to coated pits, followed by formation of coated vesicles (endosomes), and eventually delivery of particles to lysosomes. Pretreatment with cytochalasin B virtually prevented endocytosis of SCG particles, indicating active participation of the cytoskeleton. Determination of PGE2 production in parallel showed that SCG significantly stimulated PGE2 synthesis within minutes, whereas PEG-coated gold had no effect. When gold particles were coated with decomplemented serum instead of fresh serum, the stimulation of PGE2 was partially, but not completely, prevented, indicating that complement may be one, but not the only ligand responsible for enhanced PGE2 production. Stimulation of PGE2 synthesis by SCG was not dependent on actual endocytosis, as it was not altered by cytochalasin B pretreatment. Thus, surface ligand-receptor interaction may be sufficient to trigger PGE2 synthesis. The interaction between mesangial endocytosis and PGE2 production may be important for glomerular pathophysiology.

Animals↗
PubMed1987

Morphological aspects of the action of ADH.

Early studies employing biophysical techniques provided a model for ADH-induced water flow in which the number of small water-conducting channels in the outer facing membrane is increased by the hormone. With the development of new concepts and techniques in cell biology, the problem of ADH action now centers on organelle movement, fusion, endocytosis and vesicular traffic with the cell. In this review, endocytosis and vesicular traffic are discussed, and their application to the action of ADH is considered.

Animals↗
PubMed1986

Evidence that the heads of ADH-sensitive aggrephores are clathrin-coated vesicles: implications for aggrephore structure and function.

Antidiuretic hormone (ADH) induces the fusion of long tubular organelles (aggrephores) with the luminal membrane of the receptor cell, and the delivery of particle aggregates to the membrane. Water flow is believed to take place through the particles. Nothing is known about the origin of the particle aggregates, their incorporation into the aggrephores, or the possible relationship of the aggrephores to the vesicular traffic that takes place in the epithelial cell. In the present studies of the ADH-sensitive epithelial cells of the toad urinary bladder, we have found that the spherical heads of the aggrephores appear to be clathrin-coated vesicles. We propose that vesicles originating from sites such as the Golgi or the luminal membrane may be engaged in aggrephore assembly, the resupply of particle aggregates to the aggrephores, and/or the removal of aggregates, and that the aggrephores may be central points in the pattern of vesicular traffic in the cell.

Animals↗
PubMed1986

Contraction and relaxation of cultured mesangial cells on a silicone rubber surface.

Glomerular mesangial cells (MC) in culture are believed to contract or relax in response to agents such as angiotensin II and cyclic AMP. However, cells grown on glass or plastic surfaces are limited in their response to vasoactive agents because of the rigid surfaces to which they adhere; thus, interpretation of a change in shape as contraction, relaxation, or detachment is difficult. We have grown MC on a flexible silicone rubber (dimethylpolysiloxane) substrate (DMPS), and studied with sequential photographs several models of cell contraction, relaxation, and detachment. When the cells contracted, the DMPS became wrinkled; when the cells relaxed, the DMPS lost wrinkles. In contrast, if the cells detached, the sheet lost wrinkles as the cells became smaller and rounder. Angiotensin II (5 X 10(-7) M), and calcium ionophore A23187 (2 X 10(-6) M) increased wrinkles in more than 30% of cells at 22 degrees C and more than 40% of the cells at 36 degrees C. The earliest effect was visible within five to 10 minutes at 22 degrees C and within one minute at 36 degrees C and increased until 40 minutes; thereafter, the cells relaxed and wrinkles were reduced. 10(-1) M Na azide prevented the increase in wrinkles produced by angiotensin II. Seventy-two percent of the angiotensin II-treated cells whose margins could be seen in their entirety, and 78% of the calcium ionophore-treated cells showed a reduction in surface area at a time when new wrinkles were appearing or wrinkles were increasing in size. Dibutyryl cyclic AMP, a known smooth muscle relaxant, produced a decrease or loss of wrinkles in 90% of the cells, and an accompanying increase in surface area. Untreated control cells, observed in conjunction with the above series, showed little change in wrinkles. Ten percent DMSO, an actin-translocating agent, produced a reversible disappearance of wrinkles. These models of contraction and relaxation could be distinguished from cell detachment; EDTA, for example, in the presence of zero calcium, diminished both cell size and wrinkles, with an accompanying lifting of cells from the surface. Similar results were obtained with cytochalasin B and chlorpromazine. Thus, the silicone rubber system accurately reflects the contraction, relaxation and detachment of cultured mesangial cells in response to a variety of agents.

Angiotensin II↗
PubMed1985

Fusion images and intramembrane particle aggregates during the action of antidiuretic hormone. A rapid-freeze study.

Antidiuretic hormone (ADH) causes the appearance of water-conducting particle aggregates in the luminal membrane of receptor cells in amphibian bladder and skin, and in the mammalian collecting duct. The aggregates originate from cytoplasmic tubules that fuse with the luminal membrane during ADH stimulation. We have studied the process of fusion and the structure of the particle aggregates by a rapid-freeze technique that renders chemical fixation and glycerol protection unnecessary. Our findings differ in some important respects from previously published work. Aggregate particles, in our study, partition equally between the external (EF) and protoplasmic (PF) membrane leaflets, rather than remaining in the protoplasmic leaflet exclusively. By including the entire population of fusion images in our survey, we have found that aggregate delivery in ADH-treated cells proceeds preferentially from small fusion images whose diameter is significantly less than the 0.12 micron characteristic of the carrier tubules themselves. We have also found that, even in unstimulated preparations, fusion images are numerous, being mostly of small diameter. ADH stimulation produces a moderate increase in the number of fusion images and a significant increase in fusion-image diameter. These findings indicate that the individual particles are mobile within the membrane, lacking interparticle linkage. In addition, contact of cytoplasmic tubules with the luminal membrane may take place even in the absence of ADH, producing small fusion images which are not associated with aggregate delivery to the luminal membrane.

Animals↗
PubMed1985

Autoradiographic studies of solute transport across the toad bladder.

The autoradiography of diffusible, hydrophilic solutes presents special problems in localization of the labeled solute under study. We present studies of the movement of 14C-labeled urea, and 14C- and 3H-labeled sucrose across the isolated urinary bladder of the toad, a vasopressin-sensitive epithelium, using a technique that avoids exposure to water throughout all processing steps and minimizes error caused by isotope scatter. We have shown a significant increase in 14C urea entry into epithelial cells following vasopressin, and a significant decrease following phloretin, an agent that selectively blocks vasopressin-stimulated urea transport. The autoradiographic technique confirms the luminal site of action of phloretin. Studies of 14C and 3H sucrose labeling show that this molecule is virtually excluded from the cell. The current method of grain counting is capable of yielding reliable information in studies of epithelial transport.

Animals↗
PubMed1985

Evidence for cycling of aggregate-containing tubules in toad urinary bladder.

Antidiuretic hormone (ADH) promotes the fusion of cytoplasmic tubular structures with the luminal membrane of receptor tissues such as toad urinary bladder. To determine whether fusion is a continuous cyclic process, bladders were stimulated with ADH with colloidal gold in the luminal bathing medium. After as little as 15 min of stimulation, gold-filled tubules were seen in the cytoplasm, evidence that cycling was indeed taking place. Serial sections confirmed that these tubules had no connection with the luminal membrane, and had returned to the cytoplasm. Cessation of ADH stimulation, followed by a second stimulation, greatly reduced the number of gold-filled cytoplasmic tubules, suggesting that many tubules were capable of refusion. Mean fusion event diameter underwent significant changes, enlarging at 15 min, and contracting at 60 min. Thus, ADH initiates a process of continuous cycling of cytoplasmic tubules between cytoplasm and luminal membrane.

Animals↗
PubMed1985

Accidental death associated with motorized wheelchair use: a case report.

Motorized wheelchairs provide independence for individuals with a wide variety of severely disabling conditions. The benefits of powered mobility are widely appreciated. In this case report, one potential risk associated with motorized wheelchairs is illustrated by the accidental death of a young man with Duchenne type muscular dystrophy. Careful patient selection, anticipatory guidance and training should help minimize any risks.

Accident Prevention↗