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Biomedical subjects

S E Mayer

Publications and source records attributed to S E Mayer.

At least 19 recordsLinked to original sources
PubMed1994

5-Hydroxytryptamine type 2A and 2C receptors linked to Na+/K+/Cl- cotransport.

The goal of the present paper was to investigate 5-hydroxytryptamine (5HT)2A and 5HT2C receptor regulation of ion transport in fibroblast cell lines transfected with these receptors. Na+/K(+)-ATPase and Na+/K+/2Cl- cotransport were measured with 86Rb+ as a surrogate for K+ uptake. Serotonin agonists had no effect on Na+/K(+)-ATPase activity in either cell line. Bumetanide, an antagonist of Na+/K+/2Cl- cotransport, almost completely blocked ouabain-insensitive K+ uptake in both cell lines, with an IC50 of about 1 microM. 36Cl- uptake was 2-fold greater than 86Rb+ uptake, consistent with the expected 2:1 stoichiometry. In addition, the Cl-/HCO3- uptake blocker 4,4'-diisothiostilbene-2,2'-disulfonic acid had no effect on Cl- uptake. The 5HT2A/2C receptor agonist (-)-2,5-dimethoxy-4-bromoamphetamine increased ouabain-insensitive K+ uptake, and this effect was blocked by bumetanide. The receptor antagonists mianserin and mesulergine, but not spiperone, blocked (-)-2,5-dimethoxy-4-bromoamphetamine responses in fibroblasts transfected with 5HT2C receptors, and all three antagonists blocked the effects in cells expressing 5HT2A receptors. Ouabain-insensitive 22Na+ uptake was similarly affected. 5HT receptor-related actions were not observed in untransfected parent NIH/3T3 fibroblasts. Thus, we have demonstrated that 5HT2C and 5HT2A receptors are linked to activation of Na+/K+/2Cl- cotransport in transfected fibroblasts. This activity may be a factor in the pharmacological actions of 5HT agonists and antagonists.

3T3 Cells↗
PubMed1993

Sodium-dependent antiporters in choroid plexus epithelial cultures from rabbit.

The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na(+)-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO3- plus 95% O2/5% CO2. Intracellular pH (pHi) was determined from the steady-state distribution of [14C]benzoate. Recovery after acidification with NH4Cl was rapid (t1/2 = 5 min) and was dependent on external Na+ (EC50 = 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na+/H+ antiport, blocked 80% of recovery when [Na+] was 5 mM with IC50 values of 100 nM. However, neither drug blocked recovery in normal [Na+]. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl-/HCO3- antiports, blocked recovery of pHi in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na+ channel, nor with dimethylbenzamil, an inhibitor of Na+/Ca2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of 22Na+ uptake was very rapid (227 pmol/micrograms of DNA/min at pH 6.2), was dependent on external [Na+] with an EC50 value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of 22Na+ uptake by hexamethyleneamiloride was 60% with an IC50 value of 76 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Characterization of hormone-sensitive adenylate cyclase in rabbit gallbladder mucosa.

1. We have developed a plasma membrane preparation from the mucosal epithelium of rabbit gallbladder and have characterized the hormonal sensitivity of adenylate cyclase in this preparation. 2. Basal activity is low and is stimulated by GTP and GppNHp. Hormonal stimulation is largely dependent on exogenous guanine nucleotide. 3. Several prostaglandins (E1 approximately E2 greater than A1 greater than B1), vasoactive intestinal peptide and the beta-adrenergic agonist, isoproterenol, stimulate mucosal adenylate cyclase activity; a variety of peptides and neurotransmitters (secretin, cholecystokinin, arg-vasopressin, oxytocin, histamine, dopamine and serotonin) are without effect. 4. The data support the hypothesis that the inhibitory effect of prostaglandins, vasoactive intestinal peptide, and isoproterenol on gallbladder fluid absorption in certain species may be mediated by cyclic AMP. 5. The membrane preparation should be useful in further characterizing hormone receptor-transducer interactions of the gallbladder mucosal epithelium.

Adenylyl Cyclases↗
PubMed1986

Methacholine sensitivity and cAMP protein kinase in tracheal smooth muscle.

We studied regional variation in canine trachealis smooth muscle sensitivity and responsiveness to methacholine as well as basal and methacholine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase activity. The trachea between the cricoid cartilage and the carina was divided into three segments of equal length (designated cervical, middle, and thoracic regions), each consisting of approximately 12-14 cartilage rings. Smooth muscle strips from each of the three regions were exposed to cumulative half-log increments of methacholine chloride. The sensitivity (-log EC50) and responsiveness (force per cross-sectional area and force per milligram protein) of the smooth muscle to methacholine in each region was determined from these data. Smooth muscle strips from cervical and thoracic regions were frozen before and after exposure to cumulative half-log increments of methacholine up to each region's previously determined EC50. Frozen samples were assayed for cAMP content or cAMP-dependent protein kinase activity. The relationship between resting tension and methacholine sensitivity and responsiveness were studied. For the size strips we used, 4 g resting tension set the average cervical and thoracic strips at 96 and 101% of their optimal length, respectively. The methacholine EC50 was not affected by a variation in resting tension. Sensitivity to methacholine was 7.1, 6.8, and 6.5 for cervical, middle, and thoracic regions, respectively. The responsiveness of the cervical and thoracic smooth muscle to methacholine was 16.4 and 16.3 g force/mm2, respectively, at an EC50 methacholine. Basal cAMP was lower in cervical smooth muscle than in thoracic. cAMP-dependent protein kinase activity ratios under both basal and EC50 methacholine-stimulated conditions were lower in cervical smooth muscle than in thoracic. We have observed in trachealis smooth muscle an inverse relationship between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity. We suggest that cAMP and cAMP-dependent protein kinase play a role in the regulation of airway smooth muscle sensitivity to cholinergic agonists.

Animals↗
PubMed1985

Inhibitory effect of methacholine on drug-induced relaxation, cyclic AMP accumulation, and cyclic AMP-dependent protein kinase activation in canine tracheal smooth muscle.

Functional antagonism between bronchoconstricting and bronchodilating pathways was examined in canine tracheal smooth muscle. Trachealis strips were contracted with either 0.3 microM (EC55) or 3.0 microM (EC80) methacholine before being relaxed by the cumulative addition of isoproterenol, prostaglandin E2, or forskolin. The EC50 for all three relaxants was increased 10-fold in tissues contracted with 3.0 microM methacholine vs. those contracted with 0.3 microM methacholine. Moreover, contracting tissues with the higher concentration of methacholine reduced the maximum relaxation induced by prostaglandin E2 and isoproterenol. Forskolin produced total relaxation regardless of the concentration of methacholine used and thus was a much more effective bronchodilator than either isoproterenol or prostaglandin E2. The inhibitory effect of methacholine on the relaxant response to these agents was paralleled by a reduction in drug-stimulated cyclic AMP-dependent protein kinase activity. Methacholine reduced the maximum activation of cyclic AMP-dependent protein kinase elicited by isoproterenol, prostaglandin E2 and submaximal concentrations of forskolin, which was a much more powerful enzyme activator than the other two agents. The ability of a maximum concentration of forskolin (30 microM) to activate cyclic AMP-dependent protein kinase was not inhibited by methacholine. Although methacholine also appeared to suppress drug-stimulated cyclic AMP accumulation, the inhibitory effect was only statistically significant in forskolin-treated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside.

Methacholine (3 microM) and sodium nitroprusside (300 microM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 microM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0 degree C) and with an abbreviated incubation time (2.5 min). When assayed at 30 degrees C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 microM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0 degree C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.

Animals↗
PubMed1984

Effects of adrenalectomy on activation of glycogen phosphorylase in rat myocardium.

Adrenalectomy causes a depressed glycogenolytic response to catecholamines in myocardium. Total phosphorylase activity (a + b) is 20% lower in isolated, perfused hearts from adrenalectomized (ADX) rats compared with hearts from sham-operated (sham) rats even though the basal activity ratios (-AMP/+AMP) do not differ. In response to epinephrine (50 nM), the sham group has a higher activity ratio than the ADX group (0.23 vs. 0.16); the difference in specific activities of phosphorylase a in the two groups is even greater, 87 versus 49 U/mg protein. The glycogen content of the heart is 30% lower in the ADX group. Adrenalectomy does not alter the accumulation of cAMP and activation of cAMP-dependent protein kinase caused by epinephrine. Although rat heart contains a heat-stable phosphatase inhibitor, the activity of this inhibitor, as judged by phosphorylase phosphatase activity, is not altered by epinephrine stimulation or by adrenalectomy. Epinephrine perfusion increases the activity ratios (pH 6.8:8.2) of phosphorylase kinase equally in sham and ADX hearts; however, the specific activities of phosphorylase kinase (basal and hormone-stimulated) at either pH are lower after adrenalectomy. The sensitivity of phosphorylase kinase activity to stimulation by calcium is the same in the sham and ADX groups. A radioimmunoassay for phosphorylase kinase detects 10% less of this enzyme in hearts from adrenalectomized animals. Specific activities at pH 6.8 and 8.2 based on the quantity of phosphorylase kinase detected by radioimmunoassay suggest a lower phosphorylation state in the ADX group. Decreases in quantities of phosphorylase and phosphorylase kinase and enzyme dissociation due to glycogen depletion could all contribute to a depressed glycogenolytic response in the ADX group.

Adrenal Glands↗
PubMed1983

Functional antagonism in canine tracheal smooth muscle: inhibition by methacholine of the mechanical and biochemical responses to isoproterenol.

The biochemical basis for the functional interaction between bronchoconstricting and bronchodilating pathways was investigated. Contracting canine trachealis strips with increasing concentrations of methacholine resulted in a progressive shift to the right of isoproterenol concentration-response curves. Thus, the EC50 for the relaxant response to isoproterenol was nearly 500-fold higher in preparations exposed to 3.0 microM methacholine than in tissues exposed to 0.03 microM methacholine. The maximum relaxation produced by isoproterenol was also dependent upon the initial muscarinic cholinergic tone. For example, isoproterenol reversed completely the contraction induced by 0.03 microM methacholine but did not relax trachealis strips contracted with 30 microM methacholine. To identify the molecular mechanism responsible for this functional antagonism, experiments were conducted to determine the effect of methacholine on isoproterenol-stimulated cyclic AMP accumulation and cyclic AMP-dependent protein kinase activation. Methacholine did not alter basal cyclic AMP content but did reduce cyclic AMP accumulation in response to isoproterenol. Furthermore, the ability of isoproterenol to activate cyclic AMP-dependent protein kinase was inhibited by methacholine in a concentration-dependent manner. This inhibition paralleled the decrease in mechanical responsiveness to isoproterenol. These results suggest that muscarinic cholinergic stimulation of canine tracheal smooth muscle functionally antagonizes the relaxant responses to beta adrenergic agonists and that a portion of this antagonism may be due to a suppression of catecholamine-stimulated cyclic AMP accumulation and cyclic AMP-dependent protein kinase activation.

Animals↗
PubMed1982

Cyclic nucleotide-dependent protein kinases in airway smooth muscle.

Because of the potential importance of cyclic nucleotide-dependent protein kinases in the regulation of airway smooth muscle tone, we have examined some of the characteristics of these enzymes in the soluble fraction of canine trachealis homogenates. In the absence of added cAMP, the heat-stable cAMP-dependent protein kinase inhibitor (PKI) abolished only a half of the 32P incorporation into mixed histones. The remaining activity appeared to be contributed by a cyclic nucleotide-independent enzyme. Phosphotransferase activity was enhanced 5-fold by 5 microM cAMP but only 70% of the cAMP-stimulated activity could be inhibited by PKI. The sensitivity of the cyclic nucleotide-dependent, PKI-resistant enzyme to cAMP, cGMP, and Mg2+ indicated that it was cGMP-dependent protein kinase. Because of the large amount of cyclic nucleotide-independent activity, and the ability of cAMP to activate cGMP-dependent protein kinase, the traditional "-cAMP/+cAMP" ratio did not provide an accurate assessment of the in vivo activation state of cAMP-dependent protein kinase. However, a modified assay was developed which allowed the precise measurement of cAMP-dependent, cGMP-dependent, and cyclic nucleotide-independent protein kinase activities. Using this new method, the cAMP-dependent protein kinase activity ratio of 0.239 in untreated trachealis strips was increased to 0.355 and 0.386 by prior exposure of the intact tissue to the smooth muscle relaxants isoproterenol and prostaglandin E2, respectively. The results of this study are consistent with the proposed role of cAMP-dependent protein kinase in the regulation of smooth muscle contractile function.

Animals↗
PubMed1981

Regulation of guinea pig heart phosphorylase kinase by cAMP, protein kinase, and calcium.

In skeletal muscle the activation of phosphorylase kinase (PK) associated with phosphorylation of the enzyme can be measured as an increase in the pH 6.8:8.2 activity ratio. Phosphorylation leads to a 20- to 30-fold increase in PK activity (PKA) at pH 6.8 and a large decrease in the Km for phosphorylase. Perfused guinea pig hearts exposed to isoproterenol (0.3 microM) showed an increase in PKA, but without an increase in the pH 6.8:8.2 activity ratio. In a 10-fold dilution of guinea pig heart cytosol exposed to cAMP + methylisobutylxanthine, PKA was stimulated twofold at pH 7.5. Addition of exogenous protein kinase stimulated PKA fourfold. Both methods of activation were reversible and were blocked by the heat-stable inhibitor of protein kinase. Guinea pig heart PK was Ca2+-dependent requiring 0.6 microM Ca2+ for half-maximal activity. Kinetic studies indicate that the Km of guinea pig heart PK for phosphorylase b at pH 6.8 is not markedly reduced after in vitro activation (35%). This could explain the observed lack of increase in the pH 6.8:8.2 activity ratio after exposure of hearts to isoproterenol. The time course for the activation of inotropic state and the glycogenolytic pathway in perfused guinea pig hearts by isoproterenol showed that these processes were maximally activated within 25 s. However, PK remained activated for 2 min, long after the other biochemical and physiological parameters had returned to control values. These data suggest that Ca2+, not phosphorylation state, is important in regulating the return of dP/dt to control levels after beta-adrenergic stimulation.

Animals↗
PubMed1981

Isoproterenol-induced restoration of contraction in K+-depolarized hearts: relationship to cAMP.

The action of isoproterenol on inotropic state, cAMP concentration, and phosphorylase b-to-a conversion was studied under conditions that are known to alter membrane properties in guinea pig papillary muscles. In accord with the results of other investigators, contractile events induced by electrical stimulation were abolished by tetrodotoxin (10 microM) or 22 mM K+ and were subsequently restored by the addition of isoproterenol (10 nM-100 microM). However, in the presence of 22 mM K+, but not tetrodotoxin, the dose-response and temporal relationships between isoproterenol and elevations in cAMP concentration were shifted to the right, whereas those for phosphorylase activation were shifted to the left. Thus the low concentrations (less than 10 nM) of isoproterenol that restored tension development did not produce a measurable increase in cAMP. Contractile responses induced by 10 nM isoproterenol were blocked by (--)-propranolol but not by (+)-propranolol or phentolamine. Methoxamine did not restore contractile events in 22 mM K+-treated muscles or induce changes in cAMP content or phosphorylase activation. These results show that conditions can be obtained (e.g., partial depolarization of cardiac cell membranes) in which beta-adrenergic receptor activation leads to restoration of inotropic state in guinea pig myocardium without an obligatory increase in tissue cAMP content. The restoration of contractile events and enhancement of phosphorylase response under these conditions suggest that beta-adrenergic receptor activation may lead to Ca2+-channel activation directly or by increasing a small pool of cAMP and thereby altering localized protein phosphorylation in the cell membrane.

Animals↗