Modeling beta-adrenergic control of cardiac myocyte contractility in silico.

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Functional assessment of recombinant human alpha(2)-adrenoceptor subtypes with cytosensor microphysiometry.

We applied the Cytosensor Microphysiometry system to study the three human alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), expressed in Chinese hamster ovary (CHO) cells, and assessed its potential in the quantitative monitoring of agonist activity. The natural full agonist, (-)-noradrenaline, was used to define agonist efficacy. The imidazole derivative dexmedetomidine was a potent full agonist of all three receptor subtypes. The imidazolines clonidine and UK 14,304 (5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) appeared to be partial agonists at alpha(2B)-adrenoceptors (E(max) approximately 60% of (-)-noradrenaline) but full agonists at alpha(2A)- and alpha(2C)-adrenoceptors. The responses mediated by all three alpha(2)-adrenoceptor subtypes were partly inhibited by the sodium-hydrogen (Na(+)/H(+)) exchange inhibitor, MIA (5-(N-methyl-N-isobutyl)-amiloride). The agonist responses were totally abolished by pretreatment with pertussis toxin in cells with alpha(2A)- and alpha(2C)-adrenoceptors, and partly abolished in cells with alpha(2B)-adrenoceptors. The residual signal in alpha(2B)-cells was sensitive to the intracellular Ca(2+)chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester). Cholera toxin (which acts on G(s)-proteins) had no effect on the agonist responses. The results suggest that the extracellular acidification responses mediated by all three human alpha(2)-adrenoceptor subtypes are dependent on Na(+)/H(+)exchange and G(i/o) pathways, and that alpha(2B)-adrenoceptors are capable of coupling to another, G(i/o)-independent and Ca(2+)-dependent signaling pathway.

G-proteins in rat blood vessels--II. Assessment of functional involvement.

1. In this study, we compared G-protein-mediated contractile responses of rat aorta and mesenteric artery rings induced by the alpha-adrenoceptor agonist, norepinephrine (NE) and by the direct G-protein activator, sodium fluoride, using various probes. 2. Activator of the stimulatory G-protein (Gs), cholera toxin (CT), attenuated the sensitivity and maximum contractile response of both aorta and mesenteric artery to NE and sodium fluoride. The effect of the toxin on the NE-sensitivity was greater in mesenteric artery. 3. Pretreatment of tissues with the inhibitor of Gi-protein, pertussis toxin (PT), reduced the sensitivity as well as maximum contraction of both the aorta and mesenteric artery to sodium fluoride, and of the mesenteric artery to NE. PT attenuated only the sensitivity but not the maximum contraction of the aorta to NE. The inhibitory effect of PT on sensitivity to NE or sodium fluoride was greater in the aorta. 4. NE and sodium fluoride-induced contractions were reduced by the sulfhydryl G-protein inhibitor, N-ethylmaleimide (NEM) in both blood vessels. NEM produced greater inhibitory effect on the sensitivity of the aorta to both contractile agents. 5. These data demonstrate that CT, PT and NEM-sensitive G-proteins are involved in NE- and sodium fluoride-induced contractile responses of the rat aorta and mesenteric artery. The differential effects of the G-protein probes indicate that certain variations in G-protein-mediated contractile responses exist among the two blood vessels, suggesting that G-protein involvement in functional responses may vary with the type of blood vessel investigated.

Assessment of water and solute absorption from experimental hypotonic and established oral rehydration solutions in secreting rat intestine.

Water and solute absorption from three experimental hypotonic oral rehydration solutions (HYPO-ORS; sodium 45, 60 and 75 mmol/L, glucose 90 mmol/L), the World Health Organization recommended ORS (WHO-ORS; sodium 90 mmol/L, glucose 111 mmol/L), and the British National Formulary recommended ORS (BNF-ORS; sodium 35 mmol/L, glucose 200 mmol/L), have been assessed by perfusion studies in cholera toxin-induced secreting rat intestine. Net water absorption was greatest from the most hypotonic solution (HYPO-45; P less than 0.05). UK-ORS prevented net water secretion and WHO-ORS promoted moderate net water absorption. Net sodium secretion was seen with all solutions but was least from WHO-ORS and greatest with BNF-ORS (P less than 0.01). Glucose absorption was similar from BNF-ORS, WHO-ORS and HYPO-45 and in each case was greater than glucose absorption from HYPO-60 and HYPO-75 (P less than 0.05). These results suggest that net water and sodium absorption from ORS may be enhanced if osmolality is reduced by decreasing the glucose content.

Identification of the pertussis and cholera toxin substrates in normal and N-ras transformed NIH3T3 fibroblasts and an assessment of their involvement in bombesin-stimulation of inositol phospholipid metabolism.

Cholera and pertussis toxin-sensitive G-proteins were examined using specific immunological probes in wild type NIH3T3 cells and in clones of these cells containing the N-ras gene attached to a promotor where expression either was (T15+) or was not (T15-) induced. The major pertussis toxin sensitive-polypeptide had the immunological characteristics of Gi2. Two distinct forms of Gs alpha (45 and 42 kDa) were identified. Long term over-expression of p21N-ras (T15+ cells) did not alter the levels of Gi2 alpha or of Gs alpha. Pretreatment of NIH3T3 or T15 cells with either pertussis toxin or cholera toxin led to the complete in situ ADP-ribosylation of the respective G-proteins. Modification of Gi2 by pertussis toxin, however, had no inhibitory effect on the ability of bombesin to stimulate the production of inositol phosphates in any of these cells lines. Treatment of these cells with cholera toxin elicited a potent inhibition of the bombesin-stimulated production of inositol phosphates. This could be mimicked, however, by other agents which increase intracellular cyclic AMP concentrations. Cholera toxin treatment did not produce a significant alteration in the number of bombesin receptors on the cell surface. These results suggest that, in the T15 cell line, enhanced coupling of bombesin receptors to a phospholipase C-mediated hydrolysis of inositol phospholipids is either produced directly by p21N-ras or that overexpression of this gene product leads to the enhanced expression or function of a cholera and pertussis toxin-insensitive G-protein which then mediates the effect.

Cytoskeletal alterations as a parameter for assessment of toxicity.

1. Some environmental substances, drugs and pollutants affect the growth of cultured cells, and produce cytoskeletal alterations. 2. These have been used as parameters for toxicity assessment of cholera toxin and pertussis toxin in Chinese Hamster Ovary cells. 3. Cholera toxin stabilized microtubules and had no effect on microfilaments and intermediate filaments. 4. Pertussis toxin affected microfilaments but appeared to have no effect on microtubules and intermediate filaments.

A semi-automated system for the assessment of toxicity to cultured mammalian cells based on detection of changes in staining properties.

We have established a semi-automated microtiter-based system for the quantification of dye binding to cultured eukaryotic cells. This system has been applied to the quantitation of toxic activities that disrupt cell monolayers and their neutralization. We have used this background as a basis for developing a detection and characterization system for activities that do not cause such gross toxicity. A prototype system has been established based on three staining procedures which in broad terms assess cellular dehydrogenase activity, and protein, DNA, and RNA content. The activity of several agents affecting cyclic nucleotide metabolism, including cholera toxin, on the staining properties of exposed monolayers has been assessed. Several new categories of cellular response are readily discernible in this latter system indicating that biological activities may be identified on the basis of the pattern of such responses. Since microtiter based systems show considerable potential for automation, it is suggested that the further development of this approach could offer a realistic prospect for numerous forms of toxicity testing on an industrial scale.

A visual technique of chemotactic assessment for pharmacological studies.

A visual assay of chemotactic response was performed by measuring the extent of the migration of polymorphonuclear leukocytes (PMN) collected from the rat pleural cavity toward a dying cell. Various types of cells (erythrocytes, PMN, or monocytes) may be destroyed by means of an argon laser, and the process may be visualized under a phase-contrast microscope (necrotactic phenomenon). This experimental model may simulate pathological events that occur in damaged tissues, and it permits the study of cellular migration during inflammatory diseases. This method did not seem to involve chemokinetic effects, but instead represented a true measure of chemotaxis. A numeration of the migrating PMNs at t = 0 and t + 10 min after cell lysis allowed a statistical analysis and the use of this phenomenon for pharmacological studies. The modification of rat PMN chemotaxis by cholera toxin, substance P, and a synthetic muramyl dipeptide are presented as examples of the use of this technique.