Regional atherosclerotic plaque properties in ApoE-/- mice quantified by atomic force, immunofluorescence, and light microscopy.

Elucidating regional material properties of arterial tissue is fundamental to predicting transmural stresses and understanding how tissue stiffness influences cellular responses and vice versa. Atomic force microscopy (AFM) was used to measure point-wise the axial compressive stiffness of healthy aortas and atherosclerotic plaques at micron level separation distances. Cross sections of plaques were obtained from a widely used animal model of atherosclerosis (ApoE-/- mice). Median point-wise values of material stiffness were 18.7 and 1.5 kPa for the unloaded healthy wall (n = 25 specimens) and plaque (n = 18), respectively. When the healthy wall was distended uniformly during AFM testing, two mechanically distinct populations emerged from comparisons of normal cumulative distributions, with median values of 9.8 and 76.7 kPa (n = 16). The higher values of stiffness may have been due to extended elastin, which was not present in the plaques. Rather, most plaques were identified via standard and immunofluorescent histology to be largely lipid laden, and they exhibited a nearly homogeneous linear elastic behavior over the small AFM indentations. Understanding the mechanics and mechanobiological factors involved in lesion development and remodeling could lead to better treatments for those lesions that are vulnerable to rupture.

Time-dependent changes in smooth muscle cell stiffness and focal adhesion area in response to cyclic equibiaxial stretch.

Observations from diverse studies on cell biomechanics and mechanobiology reveal that altered mechanical stimuli can induce significant changes in cytoskeletal organization, focal adhesion complexes, and overall mechanical properties. To investigate effects of short-term equibiaxial stretching on the transverse stiffness of and remodeling of focal adhesions in vascular smooth muscle cells, we developed a cell-stretching device that can be combined with both atomic force and confocal microscopy. Results demonstrate that cyclic 10%, but not 5%, equibiaxial stretching at 0.25 Hz significantly and rapidly alters both cell stiffness and focal adhesion associated paxillin and vinculin. Moreover, measured changes in stiffness and focal adhesion area from baseline values tend to correlate well over the durations of stretching studied. It is suggested that remodeling of focal adhesions plays a critical role in regulating cell stiffness by recruiting and anchoring actin filaments, and that cells rapidly remodel in an attempt to maintain a homeostatic biomechanical state when perturbed above a threshold value.

Nonlinear analysis of partial dopamine agonist effects on cAMP in C6 glioma cells.

Most drugs have some efficacy so that improved methods to determine the relative intrinsic efficacy of partial agonists should be of benefit to preclinical and clinical investigators. We examined the effects of partial D(1) or partial D(2) dopamine agonists using a partial agonist interaction model. The dependent variable was the modulation of the dopamine-receptor-mediated cAMP response in C6 glioma cells selectively and stably expressing either D(1) or D(2) recombinant dopamine receptors. The dissociation constant (K(B)) and relative intrinsic efficacy (E(r)) for each partial agonist were calculated using a partial agonist interaction null model in which the effects of fixed concentrations of each partial agonist on the dopamine dose-response curve were evaluated. This model is an extension of the competitive antagonist null model to drugs with efficacy and assumes only that the log-dose--response curve is monotonic. Generally, the partial agonist interaction model fit the data, as well as fits of the independent logistic curves. Furthermore, the partial agonist K(B) values could be shared across partial agonist concentrations without worsening the model fit (by increasing the residual variance). K(B) values were also similar to drug affinities reported in the literature. The model was validated in three ways. First, we assumed a common tissue stimulus parameter (beta) and calculated the E(r) values. This provided a qualitative check on the interaction model results. Second, we calculated new relative efficacy values, E(r)(beta), using the beta estimate. Third, we calculated relative efficacy using relative maxima times midpoint shift ratios (J. Theor. Biol. 198 (1999) 347.). All three methods indicated that the present model yielded reasonable estimates of affinity and relative efficacy for the set of compounds studied. Our results provide a quick and convenient method of quantification of partial agonist efficacy. Special applications and limitations of the model are discussed. In addition, the present results are the first report of the relative intrinsic efficacy values for this set of D(2) ligands.

A method for normalizing drug responses to enhance reliability of parametric statistical tests.

Experimental constraints often require that pharmacologists study the effects of treatments upon responses elicited with a single concentration of agonist. The choice of statistical analysis, nonparametric or parametric, will depend upon whether or not the responses are normally distributed. This study uses both Monte Carlo and theoretical approaches to examine the normality assumption as it applies to drug responses. Responses measured in systems where drug sensitivities follow a lognormal distribution are generally not normally distributed. A simple transformation of responses, however, can restore normality. Researchers may find it beneficial to transform response data prior to performing t tests, analysis of variance or other parametric statistics in order to preserve the power and confidence level of the test.

Stimulus amplification, efficacy, and the operational model. Part I--binary complex occupancy mechanisms.

A simple function, developed to represent the stimulus produced at the n -th step of a multistep signaling sequence, is applied to the classic model of agonist-receptor interaction. The model indicates that the relative efficacy of two agonists can be easily estimated from three experimental measures: maximal response, EC50, and apparent dissociation constant. Efficacy ratios obtained in this manner appear statistically and mathematically equivalent to those estimated with null-based methods. Enhancement of both maximal response (vertical amplification) and potency (horizontal amplification) are demonstrated to result from the interaction between highly efficacious agonists and signal transduction mechanisms. Both properties, not just relative maxima, must therefore be examined when comparing relative efficacy. Three additional generalized stimulus-response models are developed and shown to be functionally equivalent. Increasing the complexity of the function used to represent stimulus amplification does not appear to alter the conclusions derived based on the simple model of stimulus amplification. Analysis of the results also reveals a close relationship between mechanistic and operational modes of drug action, and allows operational parameters to be given mechanistic interpretation.

Stimulus amplification, efficacy, and the operational model. Part II--ternary complex occupancy mechanisms.

To understand how deviations from simple binary occupancy affect measures of efficacy, the generalized stimulus function developed in Part I was used to examine the actions of drugs in systems where occupation of the receptor was modeled using a two-state, ternary complex, or combination of mechanisms. Amplification of drug responses can occur during formation of an active agonist-receptor complex, during generation of the initial stimulus, and during signal transduction. Expressions were derived to characterize the separate contributions of these three phases. Ideally, comparison of relative intrinsic efficacy measures differences in the ability of the agonists to convert active complex into an active stimulus. In practice, differences in the ability of the drugs to form the stimulus-generating complex may also contribute to the efficacy ratio and must be taken into consideration. Failure to adequately account for differences in occupancy can result in overestimation of the efficacy ratio. The magnitude of the difference between true and experimental measures of intrinsic efficacy may be affected by G protein concentration, by the affinity between the G protein and receptor, and (in some models) by the receptor activation constant. Provided that the dissociation constant between the G protein and receptor is of the same order of magnitude as, or lower than the receptor concentration, however, experimental estimates should provide reasonably accurate estimates of the true efficacy ratio. In agreement with previously published experimental data, total G protein level was found capable of influencing agonist maximal response, Emax, and EC50 values in all four ternary complex models. The magnitude of the changes in Emax and EC50 appear to be dependent upon the efficacy of the agonist as well as characteristics of the post-receptor stimulus sequence.Additionally, the concentration-response relations for all four ternary complex models could be reduced to a modified operational format in which the apparent dissociation constant Kapp replaced the true KA, and an apparent operational efficacy, tauapp, replaced tau. tauapp can be estimated experimentally from measurements of the Kapp and EC50, while the operational maximum, Em, may be found from the calculated tauapp and the measured Emax of the response curve. These findings support the use of direct operational model-fitting in a variety of systems, regardless of the mechanisms underlying occupancy. Values of Kapp calculated using the exact formula for [ARG] displayed an anomalous rise or discontinuity where the concentrations of total G protein equaled that of the receptor protein. This discontinuity is not observed in the estimates based on approximations to [ARG], and may explain practical difficulties in evaluating the dissociation constant under these conditions.

Analysis of stimulus-response chains using nonlinear dynamics.

Stimulus chains represent those series of coupled biochemical events through which receptor occupation is transformed into physiological or pharmacological responses. These events are commonly modeled as sets of sequential hyperbolic responses that may be treated mathematically as an iterative process. Methods used in the analysis of iterated map functions such as bifurcation analysis and graphical iteration are applied to the stimulus sequence to understand the dynamics of the process. Examples are given to illustrate determination of fixed points of the map as well as techniques for determining their stability. When present, a central repelling fixed point reveals the existence and location of a stimulus threshold, while the upper attracting fixed point determines the size of the terminal stimulus (S omega) in the signaling pathway. Estimates of S omega are used to develop equations for predicting changes in agonist EC50S at each point in the stimulus chain. Similarly, Lyapunov exponents are found to provide a link between changes in cell or tissue parameters and changes in the rate of stimulus amplification and agonist maximal response. These findings demonstrate that techniques of nonlinear dynamics when combined with biochemical studies of cellular signaling, provide useful new approaches for quantitative and qualitative analysis of drug action.

Effect of ethanol dependence on GABAA antagonist-induced seizures and agonist-stimulated chloride uptake.

The functional state of GABAA receptors during physical dependence on ethanol was evaluated in two ways. First, the ability of ethanol dependence to change the convulsant potency of GABAA antagonists microinjected into the inferior colliculus was examined. A second approach evaluated the effects of ethanol dependence on the ability of muscimol or pentobarbital to stimulate chloride uptake in rat brain vesicles. In the studies examining changes in convulsant potency, bilateral microinfusions of GABAA antagonists, bicuculline methiodide and picrotoxinin, as well as the excitatory amino acid agonist, kainic acid (used as a positive control) induced similar dose-related increases in the frequency of wild-running seizures. Ethanol dependence did not significantly change susceptibility to wild-running seizure induction by an of the convulsants, although susceptibility to the more severe, clonic seizures was significantly increased for each convulsant. This suggested that the receptor-blocking effects of GABAA antagonists responsible for inducing wild-running seizures were not selectively increased by ethanol dependence, but that spread of seizure activity responsible for clonic seizures following the initiation of wild running was generally increased. Finally, in studies examining changes in GABAA receptor-mediated chloride uptake, both muscimol and pentobarbital were found to induce concentration-dependent increases in chloride uptake in rat brain vesicles. However, responses to these drugs were not reduced by ethanol dependence suggesting that a generalized adaptive decrease in GABAA receptor function was unlikely. Together these results do not provide support for the hypothesis that the GABAA receptor-chloride channel complex is down-regulated during the development of physical dependence on ethanol.

Effects of acute and chronic ethanol treatment on pre- and postsynaptic responses to baclofen in rat hippocampus.

Interactions between the GABAB receptor and acute or chronic ethanol treatment were studied using extracellular and intracellular electrophysiological recording techniques. Bath application of the GABAB receptor agonist, (-)-baclofen (0.1-100 microM) induced concentration-dependent inhibition of extracellularly recorded dendritic excitatory postsynaptic potentials (EPSPs) in the CA1 region of hippocampal slices. Responses to baclofen were unchanged relative to control either by simultaneous application of ethanol (10-60 mM) or by previous chronic ethanol exposure. The membrane potential of CA1 pyramidal neurons was reversibly hyperpolarized an average of 5 mV by pressure ejection of baclofen (1 mM). Bath application of ethanol (30 mM) alone occasionally caused a small depolarization of resting membrane potentials in CA1 neurons but failed to increase hyperpolarizing responses to pressure-ejected baclofen. However, in slices from chronic ethanol-treated animals hyperpolarizing responses to bath-applied baclofen (10 microM) were reduced by approximately 30% relative to controls. These results suggest that GABAB-mediated responses in CA1 hippocampal pyramidal neurons are relatively resistant to the acute effects of ethanol, but that continuous exposure to ethanol sufficient to induce physical dependence may evoke an adaptive reduction in some GABAB receptor mediated responses.

Intervertebral disk mineralization in progressive ankylosis mice.

The objective of this study was to quantitate the increasing mineral content of the intervertebral disk of progressive ankylosis mice and to identify changes in serum and bone mineral levels that might occur concurrent with the rapid mineralization of the disk. Serum calcium (8.43 +/- 1.01 mg/dl vs. 7.48 +/- 2.07) and phosphorus (6.66 +/- 1.32 mg/dl vs. 5.53 +/- 2.19) levels were within normal limits. Ankylosing mice had a 5.2% decrease in water content, a 24% increase in calcium content and a 29% decrease in phosphorus content of the intervertebral disks. The magnitude of these changes increased with age. There was no significant difference in mineral or water content of the vertebral bodies. The mineral levels per gram of tissue were much greater in ankylosing disks than in the vertebral bodies. No correlation was identified between ankylosing disks and serum or bone mineral levels.

Combined effects of autoregulation and vasoconstrictors on hindquarters vascular resistance.

Relative contributions of local autoregulatory tone and vasoconstrictor tone to skeletal muscle vascular resistance were studied in anesthetized rats during hypertension produced by vasoconstrictor infusion. Rats were instrumented with a Doppler flow probe on the sacral aorta (SA) to measure blood flow and to allow calculation of vascular resistance. An occluder was placed on the SA and used to produce stepwise reductions in local perfusion pressure. Pressure-flow curves for the hindquarters were obtained in the absence and presence of elevated mean arterial pressure (MAP) produced by infusion of angiotensin II (ANG II; 50-1,247 ng.kg-1.min-1) or phenylephrine (PE; 2.5-12.4 micrograms.kg-1.min-1). Both ANG II and PE infusion increased MAP. For example, MAP was increased by ANG II from 91 to 134 mmHg and by PE from 89 to 156 mmHg. In addition, infusions of ANG II and PE produced dose-dependent rightward shifts in the hindquarters pressure-flow relationship. To examine the effect of pressure on the dose-response relationships of ANG II or PE, local perfusion pressure was adjusted to remain constant at various pressure levels that were independent of MAP during drug infusions. This produced a series of distinct dose-response curves with each curve defined by a different pressure level and with each characterized by a different maximum change in vascular resistance. If local perfusion pressure was not held constant but was permitted to increase with MAP, a compound dose-response curve was obtained in which the combined effects of the change in local pressure (i.e., autoregulation) and vasoconstrictor dose on vascular resistance could be discerned. These data demonstrate that hindquarters blood flow autoregulation continues to occur in the presence of vasoconstrictors. Consequently, autoregulatory mechanisms may be stimulated by any increase in MAP whether associated with systemic vasoconstrictor infusion or activation of neurohumoral pressor systems. The result is an amplified rise in local vascular resistance.

Potentiation by glycine of anticonvulsant drugs in maximal electroshock seizures in rats.

This study evaluated the potentiation by glycine of anticonvulsant drugs in maximal electroshock seizures in rats. Administered alone, glycine (40 mmol/kg, p.o.) induced no anticonvulsant effect or neurotoxicity. Administered together with the anticonvulsants, glycine significantly enhanced the anticonvulsant potency of phenobarbital and carbamazepine. Glycine also potentiated the anticonvulsant actions of MK-801 and diazepam but did not improve the selectivity of the drugs, as effective doses were still associated with neurotoxicity. Glycine did not potentiate phenytoin or sodium divalproate. Administration together with glycine had no significant effect on the concentrations of phenobarbital or carbamazepine in the brain. Administration together with phenobarbital had no relevant effect on the concentration of glycine in the brain but administration of glycine and carbamazepine together resulted in an increased concentration of glycine in the hippocampus and brainstem. These findings indicate a possible glycine-sensitive component in the mechanism of action of phenobarbital, carbamazepine and diazepam in maximal electroshock seizures. Although the mechanism may not be mediated by a glycine-GABA interaction, the evidence does implicate a possible interaction between glycine and anticonvulsant drugs at NMDA receptors.

Comparison of contractile responses of the guinea pig ileum longitudinal muscle to ethanol and GABAA agonists.

The guinea pig ileum myenteric plexus contains GABAA receptors linked to chloride ion channels which are pharmacologically similar to those in the central nervous system. The present study examined the reported ability of acute ethanol treatment to directly activate GABAA receptors or to increase GABAA agonist-mediated activation of the GABAA receptor in the myenteric plexus. Direct addition of ethanol to preparations of the guinea pig ileum longitudinal muscle had two effects. Immediately after ethanol (10-300 mM) was added to the tissue bath a concentration-related contractile response was observed which became maximal within 10 sec and then decayed over the next 60 sec. Contractile responses to higher concentrations of ethanol (greater than 100 mM) also were followed by a sustained reduction of longitudinal muscle tone. Contractions evoked by gamma-aminobutyric acid (GABA) and GABAA agonists, 3-aminopropane sulfonic acid (APSA) (3-100 microM) or muscimol (0.3-30 microM) developed maximally and decayed within 20 sec. Acetylcholine (0.01-10 microM) induced contractions were sustained over several minutes. Preincubation of tissue strips in ethanol (30 mM) for 1 min did not alter concentration relationships for GABA, muscimol or APSA contractile responses. Furthermore, addition of ethanol (10-100 mM) simultaneously with APSA, or 0.5, 2 or 5 min before the addition of APSA, also failed to consistently enhance contractile responses. Ethanol (30 mM) also did not alter desensitization-induced reductions in contractile responses to muscimol (3 microM) caused by preincubation of tissues with muscimol (1 microM). Finally, contractile responses to ethanol and APSA were completely blocked by atropine (0.1 microM) and tetrodotoxin (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoconstriction is amplified by autoregulation during vasoconstrictor-induced hypertension.

This study investigated the degree to which autoregulation of blood flow interacts with vasoconstrictors to determine vascular resistance. Anesthetized rats were instrumented with a Doppler flow probe on the superior mesenteric artery (SMA) to measure blood flow and for calculation of vascular resistance. An adjustable occluder was placed on the SMA to set local perfusion pressure at values independent of mean arterial pressure (MAP) even when MAP was increased by the vasoconstrictors. Infusion of angiotensin II (ANG II, 50-1,247 ng.kg-1.min-1) produced a dose-dependent rightward shift in the intestinal pressure-flow relationship and elevated MAP from 85 to 127 mmHg. Low doses of phenylephrine (PE, 2.5-12.4 micrograms.kg-1.min-1) failed to shift the pressure-flow curve but did increase arterial pressure from 83 to 102 mmHg. At higher doses (25-62 micrograms.kg-1.min-1), PE also shifted the pressure-flow curve to the right. Maintaining local perfusion pressure at different values during the infusion of ANG II or PE produced a family of dose-response curves, with each exhibiting a different maximum change in resistance. When local pressure was permitted to increase with MAP, the composite dose-response curve for resistance that was obtained reflected the influence of the rise in local pressure (i.e., auto-regulation) and vasoconstrictor dose. At low doses of PE the increase in vascular resistance was attributable solely to an autoregulatory response related to the rise in MAP and not due to the constrictor effects of PE. Thus these data indicate that the rise in MAP accompanying systemic infusion of a vasoconstrictor stimulates autoregulation to amplify the local increase in vascular resistance.

Inhibition of guinea pig ileum contractions mediated by a class of histamine receptor resembling the H3 subtype.

Segments of guinea pig ileum were isolated and placed on coaxial electrodes in organ baths filled with oxygenated Krebs' solution at 37 degrees C. Twitch responses were produced with rectangular-wave stimuli (0.5 msec; 10 Hz; 4.8-12 V) delivered in 1-sec trains every 10 sec. After addition of pyrilamine to block H1-mediated contractions, histamine and N alpha-methylhistamine (NMH) inhibited the twitch responses with EC50 values of 64 and 1.7 nM, respectively. In contrast, there was no difference in potency between histamine and NMH at contractile H1 receptors in ileum longitudinal muscle or at chronotropic H2 receptors in the atria. Impromidine blocked the inhibitory effect of NMH in a competitive manner with a KB value of 26 nM, whereas inhibition caused by gamma-aminobutyric acid or by adenosine remained unaffected by impromidine. At concentrations that completely prevented the electrically induced twitch, NMH did not alter contractions produced by exogenous acetylcholine. Inhibitory responses to NMH were unaffected by cimetidine, hexamethonium, combined alpha and beta adrenergic receptor blockade, naloxone, 8-phenyltheophylline or gamma-aminobutyric acid receptor desensitization. These data support the presence of inhibitory histamine receptors in the ileum that are pharmacologically similar to the presynaptic H3 autoreceptors found in rat cortex. The distribution of H3 receptors, therefore, may not be confined to the central nervous system and their function, as with alpha-2 adrenergic sites, may not be limited to that of an autoreceptor.

Effects of intraventricular histamine and H2 receptor antagonists on intraocular pressure.

Severe ocular hypertension has been reported in a chronic glaucoma patient following use of histamine H2 receptor antagonists for treatment of peptic ulcer. Subsequent studies, however, have failed to demonstrate a significant action of topical or intravenously administered H2 blockers on intraocular pressure (IOP) in humans. In this study, cimetidine and ranitidine were administered into the cerebral ventricles of unanesthetized New Zealand White rabbits. Both drugs caused prolonged increases in IOP at a dose of 1 umol. Maximal elevations of IOP occurred approximately 20 min after drug injections and averaged 5-8 mmHg above pre-drug values. In contrast, histamine (0.3 and 1.0 umol) produced biphasic effects on IOP when given by intracerebroventricular (i.c.v.) injection. These data suggest that central mechanisms may mediate the actions of some histamine receptor agonists and antagonists on IOP.

Pressor responses to central injection of H2 antagonists not caused by GABA blockade.

In awake rats, ranitidine was more effective than cimetidine in elevating blood pressure following intracerebroventricular (i.c.v.) injection, yet neither drug affected the hypotensive response to subsequent injections of muscimol (8.8 nmol i.c.v.). Bicuculline (0.01 nmol) microinjected into the inferior colliculus of rats caused clonic seizures whereas cimetidine (100 nmol) had no effect. The antihistamines did not prevent GABAB receptor-mediated inhibition of twitch responses in transmurally stimulated guinea-pig ileum. Ranitidine potentiated rather than inhibited GABAA receptor-mediated contractions of ileum longitudinal muscle. Cimetidine had no effect on these responses except at high concentrations (3 X 10(-4) M) which caused a slight dextral shift in the contractile response curve for GABA that may be attributed to antimuscarinic actions of cimetidine. Taken together, these data do not support the concept that the centrally mediated pressor effects of H2 antagonists are caused by GABA receptor blockade.

Ranitidine potentiates ileum contractions caused by GABA and electrical stimulation.

GABA-evoked contractions of the guinea pig ileum were significantly potentiated by the histamine H2-receptor antagonist ranitidine in concentrations above 10 microM. To help define the mechanism of this interaction, the present study compared the effects of ranitidine on contractile responses of the guinea pig ileum to GABA, acetylcholine (A Ch) and electrical stimulation of intrinsic cholinergic neurons. Ranitidine, at concentrations that potentiated responses to GABA, also potentiated contractions induced by transmural electrical stimulation. The ability of ranitidine to amplify these latter responses was antagonized by atropine. Contractile responses to exogenous A Ch, however, were unaffected by ranitidine at any concentration. These results suggest that prejunctional, rather than postjunctional mechanisms, are of primary importance in the interaction between ranitidine and GABA.