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D C Mikulecky

Publications and source records attributed to D C Mikulecky.

17 recordsLinked to original sources
PubMed1991

Effect of direct suppression of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site on the interconversion of tetrahydrofolate cofactors to dihydrofolate by antifolates. Influence of degree of dihydrofolate reductase inhibition.

An important unresolved issue in antifolate pharmacology is the basis for the observation that the major portion of cellular tetrahydrofolate cofactors is preserved after dihydrofolate reductase activity is abolished by antifolates despite the fact that tetrahydrofolate cofactor-dependent purine and pyrimidine biosynthesis ceases. This has been attributed to feedback inhibition of thymidylate synthase by dihydrofolate polyglutamates that accumulate in the presence of antifolates. This report combines network thermodynamic modeling and experimental observations to evaluate the effects of direct inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site with a potent lipophilic quinazoline antifolate PD130883 on folate oxidation in cells. Computer simulations predict and the data indicate that marked PD130883 suppression of thymidylate synthase only slows the rate but not the extent of tetrahydrofolate cofactor interconversion to dihydrofolate upon complete suppression of dihydrofolate reductase with trimetrexate. These observations are consistent with earlier studies from this laboratory with fluorodeoxyuridine inhibition at the deoxyuridylate binding site. Hence, the much weaker inhibition by dihydrofolate polyglutamates at the level of thymidylate synthase cannot account for the apparent preservation of tetrahydrofolate cofactor pools in cells and has virtually no pharmacologic significance under conditions in which antifolates completely suppress dihydrofolate reductase. The extent of interconversion of tetrahydrofolate cofactors to dihydrofolate is strongly influenced by residual dihydrofolate reductase catalytic activity. Exposure of cells to 0.1 microM trimetrexate results in only approximately 60% of maximum dihydrofolate levels achieved when dihydrofolate reductase activity is abolished. Network thermodynamic simulations predict, and experiments verify, that inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate site by PD130883, when dihydrofolate reductase is only partially suppressed (approximately 85%) with 0.1 microM trimetrexate, substantially decreases (31-47%) the net level of interconversion of tetrahydrofolate cofactors to dihydrofolate. Further computer simulations predict that under conditions in which residual dihydrofolate reductase activity persists within the cells (more than about 5%), feedback inhibitory effects of dihydrofolate polyglutamates as well as other weak inhibitors of thymidylate synthase can significantly limit the extent of net interconversion of tetrahydrofolate cofactors to dihydrofolate and produce an apparent "compartmentation phenomenon" in which tetrahydrofolate cofactor pools are preserved within the cell in the presence of antifolates. Residual dihydrofolate reductase activity cannot, however, account for the partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure to high trimetrexate or methotrexate levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Modeling intestinal absorption and other nutrition-related processes using PSPICE and STELLA.

In summary, SPICE models are constructed by translating a highly organized biological system into a network diagram by using a disciplined, systematic method for converting flows through barriers and chemical reactions into branches in a network connecting the compartments in the tissue according to the identity of the flowing entities. The first step in building a simulation model is essentially the same as the first step in learning about the method. Simple mechanisms are mastered first; and then as proficiency and understanding of the system grow, these can be connected and elaborated to produce simulations more closely approximating the real complexity of the living system. Other methods exist that may be easier to deal with initially, but often they cannot be utilized as generally as SPICE owing to their inherent limitations. One program available on Apple machines that has a high degree of user friendliness is STELLA. We will make some brief comparisons here, since STELLA is often an easier way to get started in simulation and often perfectly adequate for smaller problems.

Computer Simulation↗
PubMed1989

Folate-pool interconversions and inhibition of biosynthetic processes after exposure of L1210 leukemia cells to antifolates. Experimental and network thermodynamic analyses of the role of dihydrofolate polyglutamylates in antifolate action in cells.

Folate analogs that inhibit dihydrofolate reductase result in only partial interconversion of tetrahydrofolate cofactors to dihydrofolate with preservation of the major portion of reduced cellular folate cofactors in L1210 leukemia cells. One possible explanation for this phenomenon is that low levels of dihydrofolate polyglutamates that accumulate in the presence of antifolates block thymidylate synthase to prevent depletion of reduced folate pools. This paper correlates biochemical analyses of rapid interconversions of radiolabeled folates and changes in purine and pyrimidine biosynthesis in L1210 murine leukemia cells exposed to antifolates with network thermodynamic computer modeling to assess this hypothesis. When cells are exposed to 1 microM trimetrexate there is an almost instantaneous inhibition of [3H] deoxyuridine or [14C]formate incorporation into nucleotides which is maximal within 5 min. This is associated with a rapid rise in cellular dihydrofolate (t1/2 approximately 1.5 min), which reaches a steady state that represents only 27.9% of the total folate pool. Pretreatment of cells with fluorodeoxyuridine, to inhibit thymidylate synthase by about 95% followed by trimetrexate only slows the rate of folate interconversion (t1/2 approximately 25 min) but not the final dihydrofolate level achieved. This is consistent with computer simulations which predict that direct inhibition of thymidylate synthase by 97, 98, and 99% should increase the half-time of dihydrofolate rise after trimetrexate to 40, 60, and 124 min, respectively, but the final level achieved is always the same as in cells with normal thymidylate synthase activity. The data reflect the high degree of catalytic activity of thymidylate synthase relative to tetrahydrofolate cofactor pools in the cells and the enormous extent of inhibition of this enzyme that is necessary to slow the rate of folate interconversions after addition of antifolates. The model predicts, and the data demonstrate, that virtually any residual thymidylate synthase activity will permit the interconversion of all tetrahydrofolate cofactors available for oxidation to dihydrofolate when dihydrofolate reductase activity is abolished, but the rate of interconversion will be slowed. Additional simulations indicate that the time course of cessation of tetrahydrofolate-dependent purine and pyrimidine biosynthesis after antifolates in these cells can be accounted for solely on the basis of tetrahydrofolate cofactor depletion alone. These data exclude the possibility that direct inhibition of thymidylate synthase by dihydrofolate polyglutamates, or any other intracellular folates that accumulate in cells after antifolates, can account for the rapid but partial interconversion of reduced folate cofactors to dihydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Network thermodynamic analysis and stimulation of isotonic solute-coupled volume flow in leaky epithelia: an example of the use of network theory to provide the qualitative aspects of a complex system and its verification by stimulation.

A network thermodynamic model was developed to provide insights into the nature of isotonic solute-coupled volume flow in "leaky" epithelia, where the transepithelial volume flow is assumed to be primarily through the cellular pathway. The coupled flows of solute and volume at each membrane in this four membrane model are described by the practical phenomenological equations as developed by Kedem & Katchalsky (1958). The model contains one permeable non-electrolyte solute (s) and a fixed amount of an impermeable non-electrolyte (i) inside the cell. The cell is assumed to be capable of volume regulation under the steady-state experimental conditions simulated. A solute-pump, located in the basolateral membrane, uses feedback regulation to adjust Cs in the cell in order to maintain cell volume at or near control levels in all simulations. Model behavior is, in general, very consistent with experimental observations with respect to tonicity and magnitude of volume flow over a wide range of experimental conditions. Examination of the parameter space suggests the following important features when isotonic solute-coupled volume flow moves primarily through the cellular pathway: (1) the apical membrane reflection coefficient must be less than that of the basolateral membrane; (2) the basement membrane reflection coefficient must be small; (3) the apical membrane solute permeability and reflection coefficient are the two most "sensitive" parameters and need to vary in an inverse manner in order to maintain isotonicity when both solute and volume flows increase; and (4) relationships (1) and (3) above imply the need for at least two separate solute pathways in the apical membrane, one that is shared with volume flow and one that is not.

Biological Transport↗
PubMed1986

Exploration of apical sodium transport mechanisms in an epithelial model by network thermodynamic simulation of the effect of mucosal sodium depletion: I. Comparison of three different apical sodium permeability expressions.

A model based on that of Koefoed-Johnsen & Ussing (1958) and elaborated by Hviid Larsen (1978) and Lew et al. (1979), is designed using network thermodynamic theory and used to simulate experiments performed on epithelia. Three different expressions for the apical sodium permeability are tested for their ability to reproduce the saturation of the short-circuit current with increasing mucosal sodium concentration. Using the parameters from the previous models, the sodium entry step is shown to be the rate limiting step. If the apical sodium permeability is constant, there is no saturation of the short-circuit current with increasing mucosal sodium. The saturation of the short-circuit current is simulated with versions of the model which include a variable apical sodium permeability. The phenomenological expressions used for the variable permeabilities are those proposed by Fuchs et al. (1977) and Civan & Bookman (1982). They describe the so-called feedback effect of the mucosal and intracellular sodium concentrations.

Animals↗
PubMed1986

Exploration of apical sodium transport mechanisms in an epithelial model by network thermodynamic simulation of the effect of mucosal sodium depletion: II. An apical sodium channel and amiloride blocking.

This paper is the second part of a modeling study on apical sodium transport mechanisms in tight epithelia. In the first part (this issue) we explored three expressions for the apical membrane sodium permeability (PapNa) and showed that only a PapNa which varies as a function of sodium concentration allows simulation of the well known saturation of the short-circuit current with increasing mucosal sodium concentration. However, the ad hoc expressions used have no mechanistic interpretation. We show here that if, instead of an ad hoc expression, one includes a one-site, two-barrier sodium channel in the apical membrane, the model also simulates this saturation. In addition, the equivalent apical sodium permeability computed from the simulations appears to be very similar to the phenomenological equation used by Fuchs et al. (1977) to fit the decrease of the apical sodium permeability with increasing mucosal sodium. The apical sodium channel simulated here is thus a possible mechanism for the feedback effect of the mucosal and intracellular sodium concentrations on the apical sodium permeability. This channel also allows the simulation of the competitive inhibition of the sodium current by amiloride, and the concomitant inhibition of the apical sodium permeability.

Amiloride↗
PubMed1986

Network thermodynamic modeling of hormone regulation of active Na+ transport in cultured renal epithelium (A6).

A network thermodynamic model was developed to describe steady-state ion flows (Na+,K+, and Cl-) and related electrical events in a cultured renal epithelium (A6) derived from toad kidney. Three hypotheses for explaining the steady-state increases in short-circuit current (SCC) produced by aldosterone and/or insulin were examined using the model. Changing only the number of basolateral Na+-K+ pumps produced virtually no change in SCC and was ruled out. Changing only the number of apical Na+ channels could produce sufficient increases in SCC but presented problems in the pattern of changes produced in cell ion concentrations and therefore appeared unlikely. Changing both apical and basolateral parameters in a balanced, coordinated manner produced the maximal changes in SCC with the minimal changes in cell ion concentrations and appeared to be the "best" hypothesis. In addition, it was found necessary for tight junction permeability to increase as active Na+ transport increased under open-circuit conditions. Simulations, using these results, compared favorably with experimental data on the stimulatory effects of aldosterone and insulin, both separately and together, on active Na+ transport.

Aldosterone↗
PubMed1979

Network thermodynamic approach compartmental analysis. Na+ transients in frog skin.

We introduce a general network thermodynamic method for compartmental analysis which uses a compartmental model of sodium flows through frog skin as an illustrative example (Huf and Howell, 1974a). We use network thermodynamics (Mikulecky et al., 1977b) to formulate the problem, and a circuit simulation program (ASTEC 2, SPICE2, or PCAP) for computation. In this way, the compartment concentrations and net fluxes between compartments are readily obtained for a set of experimental conditions involving a square-wave pulse of labeled sodium at the outer surface of the skin. Qualitative features of the influx at the outer surface correlate very well with those observed for the short circuit current under another similar set of conditions by Morel and LeBlanc (1975). In related work, the compartmental model is used as a basis for simulation of the short circuit current and sodium flows simultaneously using a two-port network (Mikulecky et al., 1977a, and Mikulecky et al., A network thermodynamic model for short circuit current transients in frog skin. Manuscript in preparation; Gary-Bobo et al., 1978). The network approach lends itself to computation of classic compartmental problems in a simple manner using circuit simulation programs (Chua and Lin, 1975), and it further extends the compartmental models to more complicated situations involving coupled flows and non-linearities such as concentration dependencies, chemical reaction kinetics, etc.

Animals↗
PubMed1979

A network thermodynamic two-port element to represent the coupled flow of salt and current. Improved alternative for the equivalent circuit.

A two-port for coupled salt and current flow is created by using the network thermodynamic approach in the same manner as that for coupled solute and volume flow (Mikulecky et al., 1977b; Mikulecky, 1977). This electrochemical two-port has distinct advantages over the equivalent circuit representation and overcomes difficulties pointed out by Finkelstein and Mauro (1963). The electrochemical two-port is used to produce a schematic diagram of the coupled flows through a tissue. The network is superimposable on the tissue morphology and preserves the physical qualities of the flows and forces in each part of an organized structure (e.g., an epithelium). The topological properties are manipulated independently from the constitutive (flow-force) relations. The constitutive relations are chosen from a number of alternatives depending on the detail and rigor desired. With the topology and constitutive parameters specified, the steady-state behavior is simulated with a network simulation program. By using capacitance to represent the filling and depletion of compartments, as well as the traditional electrical capacitances, time-dependent behavior is also simulated. Nonlinear effects arising from the integration of equations describing local behavior (e.g., the Nernst-Planck equations) are dealt with explicitly. The network thermodynamic approach provides a simple, straightforward method for representing a system diagrammatically and then simulating the system's behavior from the diagram with a minimum of mathematical manipulation.

Animals↗
PubMed1978

A network thermodynamic model of salt and water flow across the kidney proximal tubule.

This network thermodynamic model of kidney proximal tubule epithelium treats coupled salt and water flow across each component membrane of the epithelium. We investigate the effects of various relative internal parameter values on the concentration of transepithelial flow, the concentrations in the cell and interspace, and the distribution of flows between cellular and paracellular routes. Best fit is obtaine if the apical and basolateral membrane reflection coefficients (or) are equal. The measured transepithelial filtration coefficient, Lp, is a function not only of the component Lps but also of the internal concentrations, or's, and permeabilities. For the given system topology (i.e., connectedness), parameters of component membranes must be within a narrow range to be consistent with experimental results. The dependence of the concentration of transported fluid on the balance between the solute pump rate and the transepithelial volume flow driving force is shown. This has implications for the effects of peritubular or lumen oncotic pressure on salt and water flow. With Appendix B of this paper and a user's guide for a circuit-simulation package (e.g., SPICE or PCAP) the reader can perform similar network analyses of transport models himself.

Biological Transport↗