Vitrification and soluble carbohydrate levels inPetunia leaves as influenced by media gelrite and sucrose concentrations.

Normal nodal segments ofPetunia hybrida were grown on Murashige and Skoog salts containing varied levels of Gelrite and sucrose. Higher concentrations of Gelrite decreased vitrification while increased sucrose concentrations promoted vitrification. Leaves of vitreous plants had higher levels of reducing sugars and sucrose but lower or undetectable levels of inositol as compared to normal plants. Normal plants on medium void of inositol have the ability to synthesize inositol and maintain levels equal to that found in plants from inositol containing media.

Peptide binding to intestinal epithelium: distinct sites for insulin, EGF and VIP.

Several peptides, including insulin, epidermal growth factor and vasoactive intestinal polypeptide bind to intestinal epithelial cells. However, it is unclear whether one binding site binds several peptides or whether separate sites exist for each peptide. These studies were designed to examine the specificity of peptide binding sites on intestinal epithelial cells. Peptide binding was measured directly with [125I]radiolabelled peptides to isolated enterocytes prepared from rabbit ileum. The characteristics of insulin and epidermal growth factor binding were similar. Both insulin and epidermal growth factor specific binding was saturable, directly correlated to cell concentration and temperature and pH dependent. The total number of insulin binding sites per cell was 4500, that for epidermal growth factor was 2280. Scatchard analysis for both peptides produced curvilinear plots. Dissociation of both peptides from the binding site was increased in the presence of their respective unlabelled peptide. However, insulin specific binding was not altered by epidermal growth factor, and epidermal growth factor specific binding was unaffected by insulin. Further, both insulin and epidermal growth factor failed to inhibit the specific binding of vasoactive intestinal polypeptide to ileal enterocytes, and vasoactive intestinal polypeptide did not inhibit insulin or epidermal growth factor specific binding. These studies demonstrate that insulin, epidermal growth factor and vasoactive intestinal polypeptide interact with three distinct membrane binding sites on the enterocyte.

Problems associated with medical treatment of peptic ulcer disease.

In the United States, the drugs most commonly used to treat peptic ulcer disease are antacids and the H2-receptor antagonists cimetidine and ranitidine. Other available agents include anticholinergics and the coating agent sucralfate. Investigational drugs such as colloidal bismuth, carbenoxolone, prostaglandins, the tricyclic compound pirenzepine, and substituted benzimidazoles are not available for use in the United States. Most of the commercially available and investigational compounds have similar efficacy; therefore the optimal drug may be the one associated with the fewest adverse effects and the most convenient dosing regimen. Cimetidine causes a small number of adverse effects, including neuropsychiatric disorders, gynecomastia, impotence, loss of libido, elevation of serum creatinine and serum transaminases concentrations, and drug interactions. Some of these reactions have been of clinical significance. Presently, there are rare reports of gynecomastia, bradycardia, inhibition of acetylcholinesterase, headache, lethargy, diarrhea, and rash in patients receiving ranitidine. Antacids can produce either diarrhea or constipation and have been associated with low serum phosphorus concentrations, and metabolic alkalosis. Anticholinergics, especially in elderly or debilitated patients, can cause central nervous system disorders, intestinal atony, or urinary retention. Sucralfate may cause constipation, diarrhea, nausea, and headache. The investigational agents have their own side effect profiles. The adverse effects of anticholinergics make them unattractive therapeutic choices, and antacids and sucralfate have inconvenient dosing requirements compared with some equally efficacious alternatives. In addition, clinical experience with sucralfate in the United States is limited. The safety record of cimetidine is admirable. As clinical experience with ranitidine increases, currently unrecognized adverse effects may be reported. However, based on current data, ranitidine is as effective as cimetidine and is associated with a lower incidence of side effects.

Serotonin-induced alteration of colonic electrolyte transport in the rat.

Serotonin, a calcium-dependent intestinal secretagogue, inhibits sodium chloride absorption in the rabbit ileum, but unlike cyclic adenosine monophosphate-dependent secretagogues, does not stimulate chloride secretion. Because bethanechol, a muscarinic cholinergic agonist that acts as a calcium-dependent, noncyclic adenosine monophosphate-mediated secretagogue, both inhibits sodium chloride absorption and stimulates active chloride secretion in the rat colon, experiments were performed with serotonin to determine the effect of serotonin on colonic ion transport. In luminal perfusion studies, intravenous infusion of serotonin significantly decreased net water and net sodium transport. In in vitro studies of sodium and chloride transport performed under short circuit conditions, serotonin decreased both net sodium and net chloride absorption and increased short circuit current (1.7 +/- 0.4, 4.0 +/- 0.6, and 2.3 +/- 0.3 mu Eq/h X cm2, respectively). The decrease in net chloride transport was significantly greater than that in net sodium absorption and the difference between these changes was equivalent to the change in short circuit current. Removal of serosal calcium inhibited all of the effects of serotonin on electrolyte transport. Tetrodotoxin, an inhibitor of neurotransmission, had no effect on serotonin-induced changes in electrolyte transport. These results demonstrate that serotonin both inhibits neutral sodium chloride absorption and stimulates active chloride secretion by a calcium-dependent process in the rat colon.

Corticosteroid alteration of active electrolyte transport in rat distal colon.

To determine the effect of corticosteroids on active transport processes, unidirectional fluxes of 22Na, 36Cl, and 42K were measured under short-circuit conditions across isolated stripped distal colonic mucosa of the rat in control, secondary hyperaldosterone, and dexamethasone-treated animals. In controls net sodium and chloride fluxes (JNanet and JClnet) and short-circuit current (Isc) were 6.6 +/- 2.2, 7.6 +/- 1.6, and 1.3 +/- 0.2 mu eq X h-1 X cm-2, respectively. Although aldosterone increased Isc to 7.3 +/- 0.5 mu eq X h-1 X cm-2, JNanet (6.9 +/- 0.7 mu eq X h-1 X cm-2) was not altered and JClnet was reduced to 0 compared with controls. Dexamethasone also stimulated Isc but did not inhibit JClnet. In Cl-free Ringer both aldosterone and dexamethasone produced significant and equal increases in JNanet and Isc. Theophylline abolished JNanet in control animals but not in the aldosterone group. Aldosterone reversed net potassium absorption (0.58 +/- 0.11 mu eq X h-1 X cm-2) to net potassium secretion (-0.94 +/- 0.08 mu eq X h-1 X cm-2). Dexamethasone reduced net potassium movement to 0 (-0.04 +/- 0.12 mu eq X h-1 X cm-2). These studies demonstrate that 1) corticosteroids stimulate electrogenic sodium absorption and 2) aldosterone, but not dexamethasone, inhibits neutral NaCl absorption and stimulates active potassium secretion. The effects of mineralocorticoids and glucocorticoids on electrolyte transport are not identical and may be mediated by separate and distinct mechanisms.

Role of calcium in the regulation of colonic secretion in the rat.

In vitro experiments were performed in rat colon to define the role of calcium in the regulation of electrolyte transport. Neither basal net sodium absorption (JNanet) nor JClnet was affected by varying serosal calcium from 0 to 3.0 mM, but both were decreased by 4.8 mM calcium. Removal of serosal calcium completely inhibited the effect of bethanechol, a muscarinic cholinergic agonist, which inhibits neutral sodium-chloride absorption in 1.2 mM calcium. In contrast, theophylline significantly decreased JNanet and JClnet both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium-free media. In 3.0 mM calcium bethanechol inhibited JCLnet significantly greater than JNanet and in 4.8 mM calcium bethanechol decreased JClnet equivalent to the increase in short-circuit current without significantly altering JNanet. We conclude that 1) high [Ca2+] inhibits net sodium and net chloride absorption; 2) the alteration of electrolyte transport by bethanechol is dependent on extracellular calcium, and the alteration of electrolyte transport by theophylline is not dependent on extracellular calcium but may be dependent on intracellular calcium; and 3) in addition to inhibition of neutral NaCl absorption, bethanechol stimulates chloride secretion.

Effect of tetrodotoxin on cholinergic agonist-mediated colonic electrolyte transport.

Muscarinic cholinergic agonists stimulate electrolyte secretion in the intestine; nicotinic agonists augment absorption. Recent studies in rabbit ileum suggest that nicotinic but not muscarinic agonists act via an intermediary neurotransmitter. These in vitro studies in rat colon were performed to determine whether tetrodotoxin (TTX), a potent neurotoxin, inhibits cholinergic agonist-induced changes in electrolyte transport. TTX did not significantly alter basal ion transport or the ion transport changes produced by either theophylline or bethanechol, a muscarinic agonist. At a concentration of 1 mM carbachol, a mixed muscarinic-nicotinic agonist, had no apparent effect on ion transport but in the presence of TTX significantly decreased net sodium absorption, similar to the effect produced by 1 microM carbachol alone. These results are consistent with the concept that in low concentrations carbachol acts predominantly as a muscarinic agonist; in high concentrations carbachol exhibits both muscarinic and nicotinic properties that are equivalent. We conclude that muscarinic agonists alter electrolyte transport by acting directly on the enterocyte and that nicotinic agonists act indirectly by stimulating the release of an intermediary neurotransmitter.

Muscarinic receptors on rat isolated colonic epithelial cells. A correlation between inhibition of [3H]quinuclidinyl benzilate binding and alteration in ion transport.

Muscarinic cholinergic agonists stimulate secretion or inhibit absorption in the large intestine both in vivo and in vitro, effects that are completely inhibited by atropine, a specific muscarinic antagonist. These studies were performed to determine if the muscarinic-induced alteration in electrolyte transport in rat colon was produced by muscarinic agonists binding directly to receptors on colonic enterocytes. We found that crude membranes prepared from rat isolated colonic epithelial cells had a specific, saturable, high affinity receptor for L-[benzilic-4,4-3H]quinuclidinyl benzilate, a potent muscarinic antagonist with an apparent dissociation coefficient of 0.56 +/- 0.11 nM and a maximum number of binding sites of 42.5 +/- 5.7 fmol/mg protein. Muscarinic antagonists inhibited L-[benzilic-4,4-3H]quinuclidinyl benzilate binding in nanomolar concentrations, and muscarinic agonists inhibited L-[benzilic-4,4-3H]quinuclidinyl benzilate binding in micromolar concentrations. In parallel studies, oxotremorine and bethanechol, muscarinic agonists, and racemic quinuclidinyl benzilate altered short-circuit current in rat colon in vitro in concentrations that were in good agreement with the concentrations that inhibited L-[benzilic-4,4-3H]quinuclidinyl benzilate binding to the crude membrane preparation. Thus, these studies demonstrate that the muscarinic-induced alteration in colonic electrolyte transport is directly related to agonist binding to a specific muscarinic receptor on the colonic epithelial cell.

Mechanism of cholinergic regulation of electrolyte transport in rat colon in vitro.

Cholinergic agonists inhibit sodium and chloride absorption in the intestine and often produce secretion. To determine the mechanism of cholinergic regulation of intestinal electrolyte transport, the effects of bethanechol on ion transport were studied in the rat colon in vitro. The addition of 1 mM bethanechol produced an initially large but short-lived increase in short-circuit current (Isc) (171 +/- 19 microA/cm2). Bethanechol decreased net sodium and net chloride absorption (2.5 +/- 0.5 and 2.9 +/- 0.9 mueq . h-1 . cm-2, respectively) and increased Isc (0.8 +/- 0.3 mueq . h-1 . cm-2) during the steady-state period. All these effects were inhibited by 1 microM atropine, which alone had no effect on ion transport. The removal of either sodium or chloride also inhibited the effect of bethanechol. cAMP content did not increase in isolated enterocytes incubated with bethanechol; however, calcium removal from the serosal bathing solution inhibited the bethanechol-induced changes in ion transport. These results indicate that cholinergic muscarinic agonists alter sodium and chloride transport in the colon by inhibiting coupled NaCl absorption by a calcium-dependent, non-cAMP-mediated process.