Large-magnitude, transient, bradycardic events in rabbits.

We propose that heart period sequences are organized similarly to sentences, with a lexicon of recurrent, similarly shaped words. These words should fulfill four criteria: universality, nonrandomness, central statistical tendencies, and specific associated physiology. Here we describe a large-magnitude, transient bradycardia (LMTB) and assess whether it constitutes a word. LMTBs were seen in 11 of 12 adult female rabbits. All shape parameters were different than those of the beat-randomized and phase-randomized surrogate sequences (P < 0.05-0.001). LMTBs were 8. 4 +/- 2.9 beats and 2.64 +/- 0.87 s long and were characterized by bradycardia of 77 +/- 49 ms over 1.09 +/- 0.49 s with a recovery to baseline over 1.56 +/- 0.61 s. The LMTBs had a slower recovery than onset in 9 of 11 rabbits and were highly peaked in 10 of 11 rabbits (P < 0.05). Scalar, magnitude, and shape parameters had values with central statistical tendencies. About 76% of LMTBs were accompanied by hypotension (mean -6.1 +/- 3.9 mmHg) that lagged 2 beats behind the onset of the bradycardia and that correlated with the bradycardia (-10.5 +/- 4.1 ms/mmHg). Thus transient bradycardic events are a distinct "word" in the lexicon of heart rate variability.

Ring and link requirements for tocainide binding to the class I antiarrhythmic drug receptor on rat cardiac myocytes.

Our purpose was to assess the structural and physicochemical determinants of the binding of tocainide and several of its homologs to the class I antiarrhythmic drug receptor associated with rat cardiac sodium channels. The homologs were chosen to assess the contributions of substituents of the aryl ring and the arylamine link on drug binding. Drug affinity was measured with a radioligand binding assay using [3H]Batrachotoxin A 20 alpha-Benzoate and freshly isolated cardiac myocytes. The affinities of the homologs were compared to determine the relationship between the affinity for the receptor and the physicochemical and structural properties of the parent drug. The contributions to the free energy of binding were determined with the Gibb's equation delta G = -RT In (1/Ki). Hydrophobic interactions are important at most sites. Meta substituents on the aryl ring and substituents on the link each interact hydrophobically with the receptor and contribute about 0.3 kcal/mol of carbon. The hydrophobic pocket near the link binding site accommodates at least six carbons. A para methoxy substituent reduces the free energy of tocainide binding by 43%. This profound reduction in the free energy of binding might be due to anomolously high aqueous solubility of alkyl aryl ethers. Longer alkoxy chains contribute 1.09 kcal/mol of carbon to the binding energy. Ortho substituents contribute little to binding specificity. These findings support a notion of a complex drug receptor with hydrophilic and hydrophobic domains that recognize specific moieties on class I antiarrhythmic drugs.

Interaction of drug metabolites with the class I antiarrhythmic drug receptor on rat cardiac myocytes.

The use of class I antiarrhythmic drugs may be complicated by the presence of active metabolites. A simple technique to predict the clinical activity of these metabolites might help with the clinical use of these drugs. We tested the hypothesis that drug metabolites bind to the class I drug receptor, but that only clinically active metabolites bind appreciably at clinically observed concentrations. Using a radioligand assay, we determined whether 13 class I drug metabolites interacted with a receptor for class I drugs associated with the cardiac sodium channel. The radioligand was [3H]batrachotoxinin A20 benzoate. All 13 metabolites bound to the drug receptor with IC50 values of 2.7 to 375 microns, and a mean Hill number of 1.0 +/- 0.3. All of the seven active metabolites (N-acetylprocainamide, mono-N-dealkyldisopyramide, 5-hydroxypropafenone, N-desisopropylpropafenone, 0-demethylencainide, 3-methoxy-0-demethylencainide and desethylamiodarone) each bound to the receptor at concentrations approaching their clinical concentrations, whereas none of the six inactive metabolites (quinidine-N-oxide, 3-hydroxyquinidine, glycinexylidide, monoethylglycinexylidide, N-demethylencainide and N,0-demethylencainide) did. Using a relationship which correlates drug IC50 values and clinically observed drug concentrations we calculated predicted clinical concentrations if the metabolites were clinically active. A predicted/observed ratio < or = 10 correlates with 100% positive and negative accuracies whether a drug metabolite has clinical activity. Thus a simple radioligand assay predicts whether class I drugs have clinical activity.

Class I antiarrhythmic drugs: allosteric inhibitors of [3H] batrachotoxinin binding to rat cardiac sodium channels.

This study assessed whether class I antiarrhythmic drugs allosterically inhibit [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTXB) binding to sodium channels on freshly isolated rat cardiac myocytes. All class I drugs tested inhibited equilibrium [3H]BTXB binding in a concentration-dependent manner. Scatchard analysis showed that disopyramide, flecainide, transcainide, lidocaine and amiodarone reduced [3H]BTXB maximum binding (Bmax) whereas procainamide, mexiletine, quinidine, quinine, tocainide, propafenone, encainide and O-demethylencainide increased [3H]BTXB KD but had little effect on Bmax. Kinetic [3H]BTXB binding assays were used to assess the mechanism by which class I drugs inhibit [3H]BTXB binding. Drugs that increase the unidirectional dissociation rate constant (k-1) of [3H]BTXB probably bind to sodium channels to which [3H]BTXB is already bound. Although all class I drugs increased the k-1 of [3H]BTXB, they did so weakly and at concentrations above the IC50 values of the drugs. Thus, drug binding to [3H]BTXB-bound channels does not appear to be the predominant mechanism underlying their ability to inhibit [3H]BTXB binding. Conversely, drugs which allosterically decrease the unidirectional association rate constant (K+1) of [3H]BTXB probably bind to channels to which [3H]BTXB is not already bound. All class I drugs decreased the k+1 of [3H]BTXB, indicating drug binding to [3H]BTXB-free channels. The estimated affinities of drugs for [3H]BTXB-free channels correlated closely with the IC50 values of these drugs (r = 0.94, P < .001), suggesting that this effect is a common and major determinant in their ability to inhibit [3H]BTXB binding. The results are discussed in light of electrophysiologic theories of class I antiarrhythmic drug action.

Intestinal mucin secretion in streptozotocin-diabetic rats: lack of response to cholinergic stimulation and cholera toxin.

In diabetic rats, intestinal mucin secretion is unusually high compared with that in normal rats. These studies demonstrate that mucin synthesis is also increased in the diabetic intestine. alpha- and beta-adrenergic agonists or antagonists did not affect mucin output in either normal or diabetic animals, suggesting that altered release in diabetes was not due to goblet cells responding abnormally to adrenergic agents. The cholinergic agonist bethanechol caused a dose-dependent and atropine-sensitive increase in mucin secretion from the normal intestine but had no effect on mucin release from diabetic tissue. Atropine alone did not reduce mucin secretion from the diabetic intestine to levels found in normal tissue. Cholera toxin caused an approximately fivefold increase in mucin output from normal rats but had no effect on mucin secretion from diabetic animals. Thus, goblet cell responses to cholinergic stimulation and cholera toxin in the diabetic intestine are markedly impaired. However, loss of cholinergic control does not appear to be responsible for altered baseline mucin secretion in diabetes.

Effects of Yersinia enterocolitica infection on rabbit intestinal and colonic goblet cells and mucin: morphometrics, histochemistry, and biochemistry.

The effects of Yersinia enterocolitica on intestinal goblet cells were investigated in New Zealand white rabbits. Animals infected with Y enterocolitica were compared with weight matched and pair fed controls. Goblet cell hyperplasia developed in the distal small intestine of infected rabbits on day 1, in the mid small intestine on day 3, and in the upper small intestine on day 6. In all regions hyperplasia persisted throughout the 14 day study. The degree of hyperplasia was greater in the distal small intestine than the upper and mid regions. Goblet cells in the proximal colon of infected animals seemed to respond as those in the distal small intestine. Thus goblet cell hyperplasia developed more rapidly and to a greater extent in the ileocaecal region where mucosal injury was most severe. These changes resulted directly from Y enterocolitica infection since goblet cell numbers did not increase in pair fed controls. Histochemically, goblet cell mucins from infected rabbits were unchanged at either six or 14 days. Biochemical analysis, however, established that purified mucins from animals on day 6 after infection were less sialylated (in the small intestine) and more sulphated (in the small intestine and proximal colon). In addition, mucins from the distal small intestine and the proximal colon seemed to contain fewer but longer oligosaccharide chains.

Transcainide: biochemical evidence for state-dependent interaction with the class I antiarrhythmic drug receptor.

The mechanism of action of the lidocaine derivative transcainide was examined using [3H]batrachotoxinin 20 alpha-benzoate, which binds specifically to and stabilizes activated states of the sodium channel. Transcainide (IC50 0.3 microM) inhibited equilibrium [3H]batrachotoxinin binding to sodium channels present on freshly isolated rat cardiac myocytes. Scatchard analysis of [3H]batrachotoxinin binding showed that transcainide both reduced maximal binding and altered the KD for [3H]batrachotoxinin binding, indicating noncompetitive, allosteric inhibition. Inhibition by transcainide of [3H]batrachotoxinin binding was reversible within 60 min. We used state-dependent [3H]batrachotoxinin binding assays to examine whether transcainide preferentially binds to activated or nonactivated sodium channels. Transcainide had little effect on the k-1 of [3H]batrachotoxinin even at concentrations 1000-fold greater than its IC50, indicating low affinity of transcainide for activated channels. However, transcainide decreased the k + 1 of [3H]batrachotoxinin at a concentration very close to its IC50 concentration for inhibiting equilibrium [3H]batrachotoxinin binding. The results are discussed in terms of a model in which transcainide inhibits [3H]batrachotoxinin binding by binding specifically to and stabilizing a nonactivated state of the cardiac sodium channel.

Effects of streptozotocin-diabetes on rat intestinal mucin and goblet cells.

Intestinal mucin and goblet cells were examined in streptozotocin-diabetic rats and age-matched controls. Mucin (tissue content and secretion) was measured using a highly specific enzyme-linked immunoassay. In contrast to the increased protein to deoxyribonucleic acid ratio, an absolute decrease was observed in the mucin to deoxyribonucleic acid ratio in mucosal homogenates of the diabetic intestine. This was not due to a loss of goblet cells as their numbers per crypt-villus unit increased in diabetic rats (in proportion to the rise in enterocyte numbers and crypt-villus length). Histochemically, goblet cell mucin was unchanged in diabetes. After a 90-min incubation of everted intestinal segments in Krebs' buffer, pH 7.4, at 37 degrees C, the amount of mucin released into the medium was the same in diabetic and control rats when expressed relative to tissue deoxyribonucleic acid. However, secreted mucin represented a significantly larger proportion of the total tissue mucin content in diabetic animals. Thus, to maintain mucin output at normal levels, the rate of mucin secretion is apparently increased in the diabetic intestine, despite (or perhaps causing) a large decrease in the tissue mucin content.

Effect of Yersinia enterocolitica on intestinal mucin secretion.

Mucin and glycoprotein synthesis and secretion were evaluated in the upper, mid, and distal small intestine and in the proximal colon of rabbits infected with Yersinia enterocolitica (YE). Infected (INF) animals were examined on day 6 and compared with pair-fed controls and unmanipulated weight-matched rabbits. Tissue mucin content in vivo and mucin secretion in vitro, measured by a specific immunoassay, were significantly elevated in all four regions of the gut of INF rabbits compared with both control groups. In vitro secretion of stored glycoprotein, prelabeled with [3H]glucosamine, was not increased in the upper and mid small intestine of INF animals but was significantly elevated in the distal small intestine and proximal colon. In vitro incorporation of [14C]glucosamine was increased in all four regions of the gut of INF rabbits, but secretion of newly synthesized [14C]glycoprotein was only significantly elevated in the distal small intestine and proximal colon. A graded response was observed down the intestinal tract of INF rabbits, with the greatest increase in mucin content, synthesis and secretion occurring in the distal small intestine and proximal colon where the morphological impact of disease is also most severe.

Rabbit intestinal and colonic mucins: isolation, partial characterization, and measurement of secretion using an enzyme-linked immunoassay.

Mucin was purified separately from the upper, mid, and distal small intestine and the proximal colon of the rabbit. The carbohydrate profiles of the three intestinal mucins were the same but differed from that of the colonic mucin, which contained less N-acetylgalactosamine but more sialic acid. All four mucins had similar polymeric structures composed of large, heterogeneous glycoprotein monomers and a smaller protein of Mr approximately 120,000, held together by disulphide bonds. The three intestinal mucins, however, were more resistant to dissociation by thiol reduction or degradation by proteolysis than colonic mucin. An antibody against a pool of the purified mucins was developed in guinea pigs and used to establish an enzyme-linked immunosorbent assay (ELISA). The antibody was highly specific for rabbit intestinal and colonic mucins, showing no cross-reactivity with nonmucin components of rabbit intestinal and colonic tissue, and very little or no reactivity with purified intestinal mucins from other species. The four purified rabbit mucins had the same affinity for the antibody and similar numbers of antigenic determinants. Antigenicity was apparently associated with the protein moeity of the mucins and was critically dependent on three-dimensional conformation, since proteolysis decreased the number of antigenic determinants while thiol reduction abolished antigen-antibody affinity. Using the ELISA, the tissue mucin content and the rate of mucin secretion were found to be significantly higher in the proximal colon that in the three regions of the intestine, which were the same.