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Biomedical subjects

M Donowitz

Publications and source records attributed to M Donowitz.

At least 109 records · Page 6Linked to original sources
PubMed1989

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter.

In rabbit ileum, Ca2+/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca2+/CaM stimulate linked Na+ and Cl- absorption. The role of Ca2+/CaM-dependent phosphorylation in regulation of the brush-border Na+/H+ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na+/H+ antiporter and Ca2+/CaM-dependent protein kinase(s) and its phosphorprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca2+, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca2+/CaM and ATP inhibited Na+/H+ exchange by 45 +/- 13%. This effect was specific since Ca2+/CaM and ATP did not alter diffusive Na+ uptake, Na+-dependent glucose entry, or Na+ or glucose equilibrium volumes. The inhibition of the Na+/H+ exchanger by Ca2+/CaM/ATP was due to an effect on the Vmax and not on the Km for Na+. In the presence of CaM and ATP, Ca2+ caused a concentration-dependent inhibition of Na+ uptake, with an effect 50% of maximum occurring at 120 nM. This Ca2+ concentration dependence was similar to the Ca2+ concentration dependence of Ca2+/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca2+/CaM/ATP-inhibition of Na+/H+ exchange was reversed by W13, a Ca2+/CaM antagonist, but not by a hydrophobic control, W12, or by H-7, a protein kinase C antagonist. We conclude that Ca2+, acting through CaM, regulates ileal brush-border Na+/H+ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.

Adenosine Triphosphate↗
PubMed1989

Two calcium channels in basolateral membranes of rabbit ileal epithelial cells.

The actions of three different types of calcium channel blockers on short-circuit current (Isc) in rabbit ileum were studied. These included the phenylalkylamines, verapamil and (l)-desmethoxyverapamil (D888); the dihydropyridines, nifedipine and nitrendipine; and the benzothiazepine, diltiazem. All of the drugs decreased Isc, a change associated with increased Na and Cl absorption. Verapamil and D888 had the largest effects. The dihydropyridine, BAY K 8644, a calcium channel activator, increased Isc and decreased Na and Cl absorption, effects not inhibited by tetrodotoxin. The phenylalkylamines had an additional effect on Isc in the presence of a maximally inhibitory concentration of the dihydropyridines, suggesting the possibility of two distinct calcium channels, one of which is the L-type voltage-activated, dihydropyridine- and phenylalkylamine-sensitive channel, and the other is a channel only sensitive to phenylalkylamines but not to dihydropyridines. [3H]nitrendipine and [3H]D888 binding to an enriched preparation of basolateral membranes from ileal epithelial cells was characterized. Each ligand bound specifically and saturably to an apparently single population of high-affinity sites with [3H]D888 having three times as many binding sites as [3H]nitrendipine. [3H]nitrendipine binding was partially inhibited by verapamil and D888 and was increased by diltiazem; whereas [3H]D888 binding was inhibited completely by verapamil but only partially by nitrendipine and diltiazem. These transport and binding studies suggest the presence of two types of Ca2+ channels in ileal epithelial cells, one of which interacts with the dihydropyridines, the phenylalkylamines, and the benzothiazepines at three different sites and the other channel that only binds the phenylalkylamines.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry.

Changes in cytosolic free Ca2+ ([Ca2+]i) in response to the secretagogue carbachol have been characterized in the human colon cancer cell line T84, a model Cl- secretory cell. In this study, [Ca2+]i was determined with the fluorescence indicator fura-2 at the single-cell level with a fluorescent microscope-imaging system. Basal [Ca2+]i in T84 cells in Ringer-HCO3 solution was 76 +/- 4 nM and was decreased by exposure to Ca2+ free solution or 25 microM verapamil. The cholinergic agonist carbachol caused a concentration-dependent rise in [Ca2+]i with a Km of 4 microM and a peak increase in [Ca2+]i of approximately 50 nM. The onset of the [Ca2+]i increase was within 3 s, occurred uniformly among cells, and peaked at 10-15 s. The increase in [Ca2+]i was heterogenous in the length of time the [Ca2+]i remained elevated above basal, and cell responses could be divided into at least two groups on that basis. Blocking the contributions of intracellular Ca2+ with dantrolene inhibited the increase in [Ca2+]i as early as could be determined, whereas blocking the extracellular contribution with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), verapamil, or nifedipine inhibited a slightly later increase in [Ca2+]i. In conclusion, the initial detectable increase in [Ca2+]i caused by carbachol is due to the release of Ca2+ from internal stores, whereas the contribution of extracellular Ca2+ occurs later and at least partially involves a nifedipine- and verapamil-sensitive process.

Atropine↗
PubMed1989

Elevated intracellular Ca2+ acts through protein kinase C to regulate rabbit ileal NaCl absorption. Evidence for sequential control by Ca2+/calmodulin and protein kinase C.

Calcium/calmodulin is involved in the regulation of basal rabbit ileal active Na and Cl absorption, but the mechanism by which elevated intracellular Ca2+ affects Na and Cl transport is unknown. To investigate the roles of the Ca2+/calmodulin and protein kinase C systems in ileal NaCl transport, two drugs, the isoquinolenesulfonamide, H-7, and the naphthalenesulfonamide, W13, were used in concentrations that conferred specificity in the antagonism of protein kinase C (60 microM H-7) and Ca2+/calmodulin (45 microM W13), respectively, as determined using phosphorylation assays in ileal villus cells. W13 but not H-7 stimulated basal active NaCl absorption. H-7 inhibited changes in Na and Cl absorption caused by maximal concentrations of Ca2+ ionophore A23187 and carbachol and serotonin, secretagogues that act by increasing cytosol Ca2+, while W13 had no effect. In contrast, neither H-7 nor W13 altered the change in NaCl transport caused by the cyclic nucleotides 8-Br-cAMP and 8-Br-cGMP. These data suggest that: (a) basal rabbit ileal NaCl absorption is regulated by the Ca2+/calmodulin complex and not by protein kinase C; (b) the effect of elevating intracellular Ca2+ to decrease NaCl absorption is mediated via protein kinase C but not by Ca2+/calmodulin; (c) the effects of protein kinase C are not overlapping or synergistic with those of Ca2+/calmodulin on either basal absorption or on the effects of increased Ca2+; and (d) neither Ca2+/calmodulin nor protein kinase C are involved in the effects of cAMP and cGMP on ileal active NaCl transport.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Pathogenesis of Shigella diarrhea. XVI. Selective targetting of Shiga toxin to villus cells of rabbit jejunum explains the effect of the toxin on intestinal electrolyte transport.

To examine the mechanism by which Shiga toxin alters intestinal water and electrolyte transport, ligated loops of rabbit jejunum were incubated in vivo with purified toxin and then studied in vivo by single pass perfusion and in vitro by the Ussing chamber voltage-clamp technique. Toxin exposure led to accumulation of water in the jejunal lumen, associated with decreased active basal NaCl absorption. Glucose- and alanine-stimulated Na absorption were also reduced, while toxin had no effect on either basal short-circuit current or the secretory response to theophylline. These observations suggest that Shiga toxin selectively inhibits NaCl absorption without significantly altering active anion secretion. To localize the cellular site of toxin action, populations of villus and crypt cells from rabbit jejunum were isolated and studied. Villus cells had a greater content of the glycolipid Shiga toxin receptor, Gb3, had more toxin binding sites than did crypt cells, and were much more sensitive than crypt cells to toxin-induced inhibition of protein synthesis. These experiments demonstrate that purified Shiga toxin inhibits jejunal fluid absorption without affecting active fluid secretion by a preferential effect on villus cells. The results suggest that this is due to the differential distribution of toxin receptors on villus compared to crypt cells.

Absorption↗
PubMed1988

Pancreatic cholera syndrome due to a vasoactive intestinal polypeptide-producing tumor: further insights into the pathophysiology.

This case report describes a patient with pancreatic cholera caused by a vasoactive intestinal polypeptide-producing pancreatic tumor. The case presents several unusual characteristics of this disease. The primary tumor was a mucinous adenocarcinoma of the pancreas. The serum vasoactive intestinal polypeptide level of 2400 pmol/L is the highest reported. At this vasoactive intestinal polypeptide level, the somatostatin analogue SMS 201-995 at doses up to 2 mg/24 h did not control the 21 L/24 h stool output. Fecal incontinence due to a manometrically documented hypotonic internal anal sphincter occurred. Using surgically created stomas, the segmental gastrointestinal fluid and sodium losses were shown to be greatest from the jejunum, whereas potassium losses from the colon and small intestine were equal. The cellular mechanism for the small intestinal potassium secretion is not known.

Adenoma, Islet Cell↗
PubMed1988

Ca2+ - and cAMP-induced protein phosphorylation in lacrimal gland basolateral membranes.

Basolateral plasma membranes play an integral role in coupling of stimulus to secretion of fluid and protein from the lacrimal gland. To determine if basolateral plasma membranes contain Ca2+- or adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent protein kinases, which could phosphorylate specific proteins important for secretion, a purified preparation of basolateral plasma membranes was prepared from rat exorbital lacrimal glands by differential and density gradient centrifugation. Phosphorylation of basolateral plasma membrane proteins was studied in the presence of [gamma-32P]ATP and was analyzed by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. Increasing the Ca2+ concentration in the presence of calmodulin stimulated phosphorylation of a 52,000-Mr peptide with a maximal increase in phosphorylation obtained at 3 and 66 microM free Ca2+. The phenothiazines trifluoperazine and promethazine inhibited phosphorylation of this 52,000-Mr peptide; 50% inhibition was obtained at 15 and 95 microM, respectively. Increasing the cAMP level from 0 to 10 microM stimulated phosphorylation of another peptide of 91,000 Mr. This effect could be reproduced by guanosine 3', 5'-cyclic monophosphate, but only at 100 microM. The cAMP concentration causing 50% of maximal phosphorylation was 0.3 microM. We conclude that lacrimal gland basolateral plasma membranes contain Ca2+/calmodulin- and cAMP-dependent protein kinases and protein substrates.

Adenosine Triphosphate↗
PubMed1988

Ca2+ channel blockers interact with alpha 2-adrenergic receptors in rabbit ileum.

An interaction between Ca2+ channel blockers and alpha 2-adrenergic receptors has been demonstrated in rabbit ileum by studying the effect of clonidine on active electrolyte transport, under short-circuited conditions, in the presence and absence of several Ca2+ channel blocking agents. Clonidine, verapamil, diltiazem, cadmium, and nitrendipine all decrease short-circuit current and stimulate NaCl absorption to different extents with clonidine having the largest effect. Exposure to verapamil, diltiazem, and cadmium inhibited the effects of clonidine on transport, whereas nitrendipine had no such effect. Verapamil, diltiazem, and cadmium, but not nitrendipine, also decreased the specific binding of [3H]alpha 2-adrenergic agents to a preparation of ileal basolateral membranes explaining the observed decrease in the transport effects of clonidine. The effective concentrations of the Ca2+ channel blockers that inhibited the effects of clonidine on transport were fairly similar to the concentrations needed to inhibit its specific binding. The displacement of clonidine by calcium channel blockers is ascribed to a nonspecific effect of these agents, although the possibility that their effects are exerted via their binding to the calcium channels is not excluded.

Animals↗
PubMed1988

Age-related loss of nongoblet crypt cells parallels decreased secretion in rabbit descending colon.

The effects of aging on active electrolyte transport and epithelial cell morphology were studied in rabbit descending colon. The rate of electrolyte transport was determined using the voltage-clamp technique, while computerized morphometric techniques were used to describe age-related and secretion-induced changes in histology. Comparison of percent water content of fecal pellets between young (2-3 mo) and mature (3.5-5 yr) animals demonstrated that mature animals had significantly decreased percent stool water. In mature animals, the total number of crypt epithelial cells was significantly less than in young animals, primarily due to the loss of crypt nongoblet cells. Crypt goblet cell number and area were not significantly changed from young to mature, while the crypt nongoblet cell number and area declined 38 and 28%, respectively, in the mature colon. Theophylline elicited dilatation of the crypt lumen. The decreased crypt nongoblet cell area correlated with a decreased theophylline-induced Cl- secretory response observed in the mature colon as measured by the change in short-circuit current (Isc). Also, a significant decrease in theophylline-stimulated active Cl- secretion was observed in mature vs. young animals in the presence of mucosal barium (5 mM) to eliminate active K+ secretion. The maximum theophylline plus barium-induced increase in Isc in young colon was 3.4 +/- 0.3 mueq.cm-2.h-1 and in mature colon 2.5 +/- 0.3 mueq.cm-2.h-1 (P less than 0.05). No significant difference in basal Isc and amiloride-inhibitable Na+ absorption was found between young and mature animals. The decreased capacity to secrete Cl- and the decrease in number and area of crypt nongoblet cells corresponds with the decrease in percent water content of mature fecal pellets.

Aging↗
PubMed1988

Regulation of the rabbit ileal brush-border Na+/H+ exchanger by an ATP-requiring Ca++/calmodulin-mediated process.

Brush-border vesicles purified from rabbit ileal villus cells were used to evaluate how Ca++/calmodulin (CaM) regulates the neutral linked NaCl absorptive process, part of which is a Na+/H+ exchanger. After freezing and thawing to allow incorporation of macromolecules into the vesicles, the effect of Ca++/CaM on brush-border Na+ uptake with an acid inside pH gradient, and on Na+/H+ exchange was determined. Freezing and thawing vesicles with 0.85 microM free Ca++ plus 5 microM exogenous CaM failed to alter Na+/H+ exchange as did the addition of exogenous ATP plus an ATP regenerating system, which was sufficient to elevate intravesicular ATP to 47 microM from a basal level of 0.4 microM. However, the combination of Ca++/CaM plus ATP inhibited Na+ uptake in the presence of an acid inside pH gradient and inhibited Na+/H+ exchange, while Na+ uptake in the absence of a pH gradient was not altered. This effect required a hydrolyzable form of ATP, and did not occur when the nonhydrolyzable ATP analogue, AMP-PNP, replaced ATP. Under the identical intravesicular conditions used for the transport studies, Ca++ (0.85 microM) plus exogenous CaM (5 microM), in the presence of magnesium plus ATP, increased phosphorylation of five brush-border peptides. These data are consistent with Ca++/CaM acting via phosphorylation to regulate the ileal brush-border Na+/H+ exchanger.

Adenosine Triphosphate↗
PubMed1987

Effects of Ca2+, theophylline and promethazine on protein phosphorylation in intact cells of rabbit ileum. Correlation with active Na and Cl absorption.

The effects of Ca2+, theophylline and promethazine on the phosphorylation of microvillus membrane proteins have been studied in rabbit ileal epithelial cells, using intact cell phosphorylation techniques followed by purification of microvillus membranes, separation of peptides by two-dimensional polyacrylamide gel electrophoresis, and quantitation of phosphorylation by computerized densitometry of autoradiograms. The Ca2+ ionophore A23187 caused increased phosphorylation of four and possibly five polypeptides; theophylline increased phosphorylation of three peptides, two of which had the same Mr and pI values as the peptides altered by the Ca2+ ionophore; promethazine decreased the phosphorylation of one of the peptides increased by Ca2+ ionophore. The phosphorylated peptides, which respond similarly to more than one agent which affect ileal Na and Cl absorption, could be involved in the regulation of NaCl absorption either as transport proteins or regulators of transport proteins.

Animals↗
PubMed1987

Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles.

High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2,000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control; with both techniques, the concentration of dextran after being taken up into the vesicles was similar to that in the incubation medium, suggesting attainment of equilibrium. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca2+-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with [32P]ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport.

Adenosine Triphosphate↗
PubMed1986

The transport of an intact oligopeptide across adult mammalian jejunum.

The passage of an intact nonapeptide across adult rabbit jejunum mounted in an Ussing Chamber is demonstrated with an HPLC system which resolves the renin inhibitor Pro-His-Pro-Phe-His-Leu-Phe-Val-[3H]Phe from all labelled proteolytic cleavage products. Permeability of the peptide (0.016 cm hr-1) is approximately one-seventh that observed for the actively transported 3-0-methyl glucose (0.104 cm hr-1). Flux of the peptide is not changed by the absence of sodium. This study shows that adult mammalian intestine can transport intact oligopeptides and suggests that it will be feasible to develop orally active peptide drugs.

Animals↗
PubMed1986

Effects of calcium antagonist TMB-8 on active Na and Cl transport in rabbit ileum.

The effects of 3,4,5-trimethoxybenzoate 8-(N,N-diethylamino)octyl ester (TMB-8), an agent that traps calcium within intracellular stores, were studied on active electrolyte transport in rabbit ileum under basal conditions and after altering transport by increasing the intracellular cAMP content or by exposure to two agonists that act by altering intracellular Ca2+ (carbachol and serotonin). TMB-8 decreased the ileal short-circuit current and increased active Na and Cl absorption by increasing the mucosal-to-serosal Na and Cl fluxes. These effects were reversed by increasing the bathing solution Ca2+ to 4 mM, a concentration that itself did not alter basal ileal transport. The maximum glucose- and amino acid (alanine)-induced increase in Na absorption in the ileum was not affected by TMB-8. The effects on basal transport of TMB-8 were not associated with a change in 45Ca2+ entry across the ileal serosal surface. TMB-8 did not alter cAMP-induced secretion, as judged by its lack of effect on the increase in short-circuit current caused by 8-bromo-cAMP (10(-4) M). TMB-8 totally prevented the transport effects of carbachol but did not inhibit the effects of serotonin. These data suggest a role for intracellular Ca2+ in regulation of basal ileal Na and Cl transport but not in cAMP-induced secretion. There appear to be several pools of intracellular Ca2+ involved in neurohumoral effects on active electrolyte transport.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Effects of phorbol esters on sodium and chloride transport in rat colon.

To determine the role of protein kinase C in the regulation of active electrolyte transport in rat descending colon, the effects of phorbol dibutyrate (PDB) were studied using the Ussing chamber/voltage-clamp technique. PDB added to the serosal surface increased the short-circuit current in a concentration dependent manner with a EC50 of 3 X 10(-8) M and a maximal effect at 10(-7) M PDB. The effect was not seen with the inactive alpha-phorbol analogue but was reproduced with 1-oleoyl-2-acetylglycerol, a more permeable analogue of diacylglycerol. PDB caused a decrease in mucosal-to-serosal and net fluxes of Na and Cl and an increase in serosal-to-mucosal Cl flux, indicating inhibition of Na and Cl absorption and stimulation of Cl secretion. The PDB-induced increase in Cl secretion was virtually abolished by both indomethacin and ibuprofen, indicating a dependence on arachidonic acid metabolism via the cyclooxygenase pathway. The Cl secretion was inhibited by verapamil and Ca2+-free bathing solution on the serosal surface but not by dantrolene, suggesting the importance of extracellular Ca2+ but not intracellular stored Ca2+ in the PDB-induced secretion. The Cl secretory effect was also inhibited by tetrodotoxin and atropine, suggesting involvement of cholinergic nerves. In contrast, the PDB-induced decrease in Na and Cl absorption was not dependent on metabolites of the cyclooxygenase pathway, not dependent on extracellular Ca2+, and not blocked by tetrodotoxin. It appears likely that protein kinase C is involved in the regulation of rat colonic active Na and Cl absorption and electrogenic Cl secretion but that the pathways involved are different in the two transport systems.

Animals↗