Biomedical subjects
Publications and source records attributed to S D Levine.
Diuretics are a mainstay of modern medical therapy. Their effects on electrolyte excretion can largely be predicted from knowledge of their sites of action along the renal tubule. This article examines the role of diuretics in cardiac and noncardiac disease.
Mercurial reagents inhibit the water permeability of erythrocytes and proximal renal tubule. We examined the effect of two such agents on vasopressin-induced water transport across toad urinary bladder. Water flows were measured in unfixed tissues and in tissues fixed either with N-ethylmaleimide (NEM) or with glutaraldehyde. When added concurrently with 20 mU/ml vasopressin, 1 mM mucosal p-chloromercuribenzene-sulfonic acid (p-CMBS) inhibited water flow within 1 h. p-CMBS also inhibited flow in tissues that had been fixed with mucosal NEM after stimulation with vasopressin. However, p-CMBS did not affect flow in glutaraldehyde-fixed tissues. In contrast, HgCl2 inhibited water flow and urea permeability even in tissues that had been fixed with glutaraldehyde after stimulation with vasopressin. Inhibition was more pronounced when HgCl2 was added to the mucosal rather than the serosal bathing medium and was not reversed by dithiothreitol. HgCl2 did not diminish the frequency or area of luminal membrane aggregates observed by freeze-fracture electron microscopy. HgCl2 also did not affect amphotericin-induced water permeability in glutaraldehyde-treated tissues, suggesting that it did not diminish the permeability of cellular barriers to flow. Our results parallel closely those reported by other investigators for water flow across erythrocytes and proximal renal tubule and suggest that mercurial reagents can directly block the vasopressin-induced water channel. The water channel at the apical membrane of the toad bladder may prove to share structural similarity with that constantly present in erythrocytes and proximal renal tubule.
Although it is well accepted that vasopressin (ADH) increases the permeability to water of the toad bladder granular cell's luminal membrane, recent studies have suggested that regulation also takes place at an additional "postluminal" site within the epithelial granular cell. These studies are based upon the observation that a number of experimental maneuvers can alter tissue permeability to water, but do not change the number of particle aggregates observed on the protoplasmic face of the granular cell's luminal membrane with freeze-fracture electron microscopy. These aggregates are believed by many investigators to mediate the transport of water across the luminal membrane. The dissociation between permeability and aggregate frequency described above has been variously interpreted as the consequence of changes in the permeability of the aggregates themselves, or of changes in the permeability of a "postluminal" barrier that is functionally in series with the luminal membrane. We attempted to distinguish between these 2 possibilities by studying paired toad bladders during 3 protocols that alter vasopressin-stimulated water flow across the intact tissue without altering aggregate frequency. Estimates of the permeability of postluminal barriers were obtained by exposing the luminal surface to amphotericin B, an antibiotic that forms water-permeant channels in the luminal membrane. Of the 3 protocols, only diminishing bladder filling volume decreased the water flow elicited by luminal amphotericin B, suggesting that only that protocol indeed decreased the permeability of some postluminal barrier. The other 2 protocols, increasing PCO2 and repeatedly stimulating the bladder with vasopressin, did not alter amphotericin B-elicited flow, suggesting that postluminal barriers were not altered by these 2 protocols.(ABSTRACT TRUNCATED AT 250 WORDS)
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Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. We examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. "High dose" cycloheximide (200 micrograms/ml) inhibited flow immediately. "Low dose" cycloheximide (1 microgram/ml) did not affect initial flow; however, flow was inhibited by the fourth restimulation. On further rechallenge, inhibition persisted but did not increase. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio (-cAMP/+cAMP), an index of in vivo cAMP effect, was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. Cycloheximide inhibited flow when 10 microM forskolin or 0.2 mM 8-BrcAMP was substituted for vasopressin in the fourth period; however, MIX (4 mM)-stimulated flow was enhanced by 1 microgram/ml cycloheximide but inhibited by 200 micrograms/ml cycloheximide. [14C]urea permeability was not inhibited by cycloheximide. Puromycin (0.5 mM) also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Some critical level of calcium appears to be necessary for maximum expression of the hydroosmotic effect of vasopressin: both maneuvers which elevate cell calcium or activate calcium-dependent enzymes and maneuvers which diminish cell calcium or inhibit its effect diminish vasopressin-stimulated water flow. It would thus appear that cell calcium might well be maintained at some optimum level for mediating vasopressin-stimulated water flow across epithelia. At the same time, there is considerable evidence supporting vasopressin-elicited alterations in cell calcium and phospholipid metabolism; however, the linkage between these alterations and any eventual effects on water permeability has not yet been established with confidence.
The synthesis, cardiac fraction III cyclic nucleotide phosphodiesterase (PDE-III) inhibition, and positive inotropic activity of a series of 2(1H)-quinazolinones are reported. A general synthesis of the series involved the cyclization of 2-aminoacetophenones with potassium cyanate in acetic acid. Modifications at the 4-position of the quinazoline nucleus were best achieved by formation of the intermediate N1-acyl-N3-phenylurea from the substituted phenyl isocyanate and appropriate carboxamide. PPA was used to ring close to the quinazoline product. Generally the SAR for the series paralleled the five-point model previously published for PDE-III inhibition. The most active analogue of the series was 5,6-dimethoxy-4-methyl-2(1H)-quinazolinone (1) (ORF 16600), which had about twice the intravenous potency of amrinone. Compound 1 is currently under development as an orally active cardiotonic.
We have reported that Pf/Pd(w) (the ratio of osmotic and diffusional water permeabilities) for the luminal membrane of toad urinary bladder is approximately 17 for tissues stimulated with either vasopressin or 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). In a recent abstract, Kachadorian and co-workers have shown that tissues stimulated with adenosine 3',5'-cyclic monophosphate (cAMP) or forskolin have a lower Pf than would be anticipated from the frequency of aggregates visualized on the flat portion of the luminal membrane using freeze-fracture electron microscopy. We report here measurements of Pf/Pd(w) for the luminal membrane of tissues receiving these agents: Pf/Pd(w) for submaximally stimulated tissues was the same, regardless of whether the stimulant was vasopressin (12.7 +/- 0.3), forskolin (13.7 +/- 0.9), or cAMP (12.0 +/- 1.3). The calculated Pd(w)'s for the series barrier were also identical (6.8 +/- 0.5, 6.5 +/- 0.3, and 8.2 +/- 1.0 X 10(-4) cm/s respectively). Our data, taken together with those of Kachadorian et al. are consistent with a number of possibilities: because our methodology does not permit estimation of Pf for the series barrier, we cannot rule out the possibility of a "post-luminal barrier" that is rate-limiting for Pf, but not for Pd(w) in forskolin- and cAMP-stimulated tissues, Pf and Pd(w) of the luminal surface aggregates could decrease in parallel, so that luminal membrane Pf/Pd(w) remains constant, and there could be a diminished frequency of fused aggregate-rich aggrephores, but not of aggregates that are on the flat portion of the luminal membrane. Only the latter can be unequivocally quantitated using freeze-fracture electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
Cholera toxin (CT) irreversibly ADP-ribosylates and activates the nucleotide-stimulatory (Ns) subunit of adenylate cyclase in many tissues, thereby eliciting cyclase-dependent functions. Although earlier studies performed at room temperature could not demonstrate CT-stimulated water transport in toad urinary bladder, subsequent work in other tissues has emphasized the need for incubation at 35-37 degrees C to effect ribosylation and the subsequent physiological effects. We found that incubating tissues with amphibian culture media, rather than Ringer solution, maintained tissue viability at this higher temperature and permitted prolonged incubation with CT. At 37 degrees C, in the presence of 0.1 mM phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine, MIX), 0.2-200 nM mucosal CT caused a dose-dependent but submaximal enhancement of water flux and urea transport. Elimination of MIX from the bath diminished subsequent CT-induced stimulation, supporting a role for adenosine 3',5'-cyclic monophosphate (cAMP) as mediator of the CT effect. The increased water flow was stable for greater than 1 h after removal of CT from the bath, consistent with irreversible stimulation of the cyclase. Mucosal CT stimulated transport to a greater degree than serosal CT, paralleling the pattern seen in the intestine, which is compatible with passage of the toxin's a subunit across the cell to the serosal membrane cyclase. Exposure of the tissue's mucosal surface to GM1 ganglioside, (the natural receptor for the CT b subunit) yielded maximal stimulation of water flow and near-maximal urea transport, presumably by increasing CT's binding to the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
In addition to cyclooxygenase and lipoxygenase pathways, the kidney can also metabolize arachidonic acid by a NADPH-dependent cytochrome P-450 enzyme to epoxyeicosatrienoic acids (EETs); furthermore, 5,6-EET has been shown to alter electrolyte transport across isolated renal tubules. We examined the effects of three EETs (5,6-, 11, 12-, and 14,15-EET) on osmotic water flow across toad urinary bladder. All three EETs reversibly inhibited vasopressin-stimulated osmotic water flow with 5,6- and 11,12-EET being the most potent. The effects appeared to be independent of prostaglandins. EETs inhibited the water flow response to forskolin but not (with the exception of 11,12-EET) the response to adenosine 3',5'-cyclic monophosphate (cAMP) or 8-BrcAMP, consistent with an effect on cAMP generation. For 11,12-EET the question of an additional inhibition at a site beyond or independent of cAMP has to be considered. To determine whether these effects were due to the EETs or to products of their metabolism, we examined the effects of their vicinal diol hydrolysis products, the dihydroxyeicosatrienoic acids. Nonenzymatic conversion of labeled 5,6-EET to its vicinal diol occurred rapidly in the buffer, whereas 11,12-EET was hydrolyzed in a saturable manner only when incubated in the presence of bladder tissue. The dihydroxyeicosatrienoic acids formed inhibited water flow in a manner paralleling that of the EETs. Both 5,6-EET and 11,12-EET (10(-5) M) prevented the increase in intracellular cAMP content observed in control tissues after vasopressin stimulation. Finally, 11,12- and 14,15-dihydroxyeicosatrienoic acid inhibited vasopressin- and forskolin-stimulated adenylate cyclase in the same rank order as their inhibition of water flow.(ABSTRACT TRUNCATED AT 250 WORDS)
The synthesis of and guinea pig contragestational screening data for several oxepane and 3,8-dioxabicyclo[3.2.1]octane analogues of zoapatanol (1) are described and their structure-activity relationships discussed. Conversion of the 5-keto group on the nonenyl side chain of 1 into a hydroxyl function enhanced the potency. Further significant enhancement in the potency was realized with the transformation of several oxepanes into the 3,8-dioxabicyclo-[3.2.1]octane-1-acetic acid derivatives. Detailed, comparative contragestational evaluation of the three most potent compounds 9, 33, and 37 is presented, which led to the selection of 33 (ORF 13811) for further biological evaluation.
The action of vasopressin (AVP) in transporting epithelia is mediated by cyclic AMP(cAMP), whereas its effects in hepatocytes are mediated by calcium and phosphoinositides. Based on our recent observation that AVP stimulates phosphoinositide turnover in toad bladder, we examined the role of calcium-phospholipid-dependent kinase (protein kinase C) as a modulator of AVP's hydroosmotic effect. Phorbol myristate acetate (PMA), which can substitute for diglyceride as an activator of protein kinase C, the diglyceride dioctanoylglycerol, and RHC-80267, a glyceride lipase inhibitor that should increase diglyceride levels, inhibited AVP-stimulated water flow, but not water flow stimulated by cAMP, suggesting inhibition of cyclic AMP production. Both the dioctanoylglycerol and RHC-80267, but not PMA, also decreased water flow in response to 8-bromo cAMP indicating a potential inhibition at post-cAMP events as well. PMA increased prostaglandin synthesis; however, inhibition of water flow persisted even when prostaglandin synthesis was completely blocked by incubation with naproxen. Furthermore, water flow was not inhibited by incubation with the inactive diglyceride substitute phorbol didecanoate, supporting the specificity of the PMA inhibition. Consistent with the site of action at adenylate cyclase suggested by the transport experiments, PMA and RHC-80237 decreased both cell cAMP content and the cyclic AMP-dependent kinase ratio (-cAMP/+cAMP), an index of intracellular cyclic AMP effect. Assay for protein kinase C activity in toad bladder epithelial cell supernatant demonstrated that the toad bladder indeed contains a kinase stimulable by phospholipid, calcium, and PMA. As an apparently independent effect, we found that addition of PMA, but not dioctanoylglycerol or RHC-80267, to the mucosal bath increased both water permeability and the frequency of granular cell luminal membrane aggregates in the absence of vasopressin, consistent with stimulation of fusion events at the luminal membrane. Our data suggest that protein kinase C can modulate AVP-stimulated water flow in toad bladder by inhibiting cAMP generation, and perhaps post-cAMP steps as well, and support the hypothesis that AVP-stimulated turnover of membrane phosphoinositides antagonize the effects of AVP via changes in diglyceride, calcium, and protein kinase C.
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Antidiuretic hormone (ADH) induces a large increase in the water permeability of the luminal membrane of toad urinary bladder. Measured values of the diffusional water permeability coefficient, Pd(w), are spuriously low, however, because of barriers within the tissue, in series with the luminal membrane, that impede diffusion. We have now determined the water permeability coefficient of these series barriers in fully stretched bladders and find it to be approximately 6.3 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of approximately 400 microns. On the other hand, the permeability coefficient of the bladder to a lipophilic molecule, hexanol, is approximately 9.0 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of only 100 microns. The much smaller hindrance to hexanol diffusion than to water diffusion by the series barriers implies a lipophilic component to the barriers. We suggest that membrane-enclosed organelles may be so tightly packed within the cytoplasm of granular epithelial cells that they offer a substantial impediment to diffusion of water through the cell. Alternatively, the lipophilic component of the barrier could be the plasma membranes of the basal cells, which cover most of the basement membrane and thereby may restrict water transport to the narrow spaces between basal and granular cells.
Using the methods described in the preceding paper (Levine et al., 1984) for measuring the magnitude of the water-permeable barriers in series with the luminal membrane, we correct measured values of Pd(w) in bladders stimulated with low doses of antidiuretic hormone (ADH) or 8-bromo cyclic AMP to obtain their true values in the luminal membrane. Simultaneously, we also determine Pf. We thus are able to calculate Pf/Pd(w) for the hormone-induced water permeation pathway in the luminal membrane. Our finding is that Pf/Pd(w) approximately equal to 17. Two channel models consistent both with this value and the impermeability of the ADH-induced water permeation pathway to small nonelectrolytes are: (a) a long (approximately equal to 50 A), small-radius (approximately equal to 2 A) pore through which 17 water molecules pass in single-file array, and (b) a shower-head-like structure in which the stem is long and of large radius (approximately equal to 20 A) and the cap has numerous short, small-radius (approximately equal to 2 A) pores. A third possibility is that whereas the selective permeability to H2O results from small-radius (approximately equal to 2 A) pores, the large value of Pf/Pd(w) arises from their location in the walls of long tubular vesicles (approximately 2 micron in length and 0.1 micron in diameter) that are functionally part of the luminal membrane after having fused with it. Aggregate-containing tubular vesicles of these dimensions have been reported to fuse with the luminal membrane in response to ADH stimulation and have been implicated in the ADH-induced hydroosmotic response.
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A role for transmembrane calcium movement in vasopressin stimulation of its target cell has been postulated based on studies with calcium entry blockers such as verapamil. We examined the effect of three sets of structurally different calcium blockers--D600 (an analogue of verapamil), diltiazem, and nifedipine--on water flow in toad bladder. D600 (200 microM), diltiazem (200 microM), and nifedipine (60 microM) inhibited vasopressin-induced water flow but enhanced adenosine 3',5'-cyclic monophosphate (cAMP)-induced water flow, suggesting that the drugs inhibit cAMP generation in response to vasopressin but enhance the response to exogenous cAMP by inhibiting phosphodiesterase activity. In the case of vasopressin stimulation, inhibition of cAMP generation appears to be the overriding effect. This was confirmed by measurements of cAMP content and the protein kinase ratio (-cAMP/+cAMP), which were significantly lower in bladders receiving both D600 and vasopressin than in those receiving vasopressin alone. Furthermore the drugs inhibited activation of adenylate cyclase by vasopressin in cell homogenates and inhibited phosphodiesterase in both homogenates and membrane-free supernatants. Thus these "calcium channel blockers" can directly alter cAMP metabolism in settings where movement of calcium should be irrelevant. The close correlation between the biochemical and transport effects of these agents suggests that their effect on water flow may occur by a direct effect on cellular enzymes or the membranes in which they reside and not by altering local calcium concentrations.