Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Interactions between responses mediated by activation of adenosine A2 receptors and alpha 1-adrenoceptors in the rabbit isolated aorta.

1. This paper describes aspects of the functional antagonism between the responses mediated by activated alpha 1-adrenoceptors and adenosine A2 receptors in the adventitia- and endothelium-denuded aorta of the rabbit. 2. Adenosine A2 receptor agonists relaxed aortic rings pre-contracted with phenylephrine. The relaxation response was agonist concentration-dependent and saturable. The respective contractile and relaxation responses were stable, reproducible, and reversible. 3. Increasing the phenylephrine concentration caused a progressive attenuation of the action of adenosine A2 receptor agonists, consisting of a decreased maximal response and a dextral shift of the adenosine agonist concentration-response curve. This functional antagonism could be completely reversed upon removal of adenosine by either the addition of adenosine deaminase or by wash-out of the adenosine agonist from the tissue. The relaxation response to the adenosine A2 receptor partial agonists, N6-cyclohexyladenosine and R-(-)-N6-(2-phenylisopropyl)adenosine, was abolished at higher phenylephrine concentrations (e.g. 30 EC50). 4. A 1000 fold increase in the adenosine concentration was required to shift the value of the EC50 of phenylephrine six fold, while a similar increase in the value of the EC50 of adenosine could be elicited by only a 32 fold increase in the phenylephrine concentration. A 30 fold increase in the phenylephrine concentration shifted the value of the EC50 of 5'-N-ethylcarboxamidoadenosine four fold. 5. Analysis of the functional antagonism between the responses mediated by these receptors using the Black & Leff (1983) operational model of agonism allowed for the estimation of the agonist dissociation constant, KA, and the apparent efficacy, tau, for both phenylephrine and adenosine A2 receptor agonists. Increasing the concentration of phenylephrine reduced the value of tau for adenosine agonists in a concentration-dependent and saturable manner. Similarly, increasing the concentration of adenosine reduced the value of tau for phenylephrine in a concentration-dependent and saturable manner. The phenylephrine KA value obtained by the method of functional antagonism (1.9 microM) was similar to that obtained by the receptor inactivation method (2.1 microM). 6. Partial occlusion of the alpha 1-adrenoceptor by the alkylating agent, dibenamine, demonstrated that the magnitude of the adenosine A2 receptor-mediated relaxation was inversely proportional to the number of functional alpha 1-adrenoceptors. 7. It is concluded that the magnitude of functional antagonism is proportional to the stimulus elicited through either receptor. We propose that this tissue preparation and pair of receptors is a good model to study quantitative aspects of functional antagonism between activated receptors.

Kinetic characterization of adenosine A2 receptor-mediated relaxation in isolated rabbit aorta.

Previous studies in our laboratory (Wiener et al., 1991, Soc. Neurosci. Abstr. 17, 989) have addressed aspects of the functional antagonism between the responses mediated by activated adenosine A2 receptors and alpha 1-adrenoceptors in adventitia- and endothelium-denuded rabbit thoracic aortic rings by steady-state protocols which ignore the time course of response generation. In the present communication we describe aspects of the time-dependent kinetics of relaxation responses to adenosine A2 receptor agonists in tissues pre-contracted with the alpha 1-adrenoceptor agonist phenylephrine. The results were analyzed by application of the model originally developed by Keitz et al. (1990, J. Pharmacol. Exp. Ther. 255, 650) to describe the relaxation response, to a beta-adrenoceptor agonist, as a first-order exponential decrease in tissue tension over time to estimate the apparent rate constant for relaxation (krel) and the magnitude of relaxation at equilibrium. The magnitude of the relaxation responses to adenosine, N6-cyclohexyladenosine, N6-methyladenosine, 5'-N-ethylcarboxamidoadenosine, and R(-)-N6-(2-phenylisopropyl)adenosine were agonist concentration-dependent and saturable, as were the apparent rate constants for relaxation. In addition, the magnitude of the apparent rate constants for relaxation and the relaxation responses were inversely proportional to the fractional occupancy of the alpha 1-adrenoceptor. The hypothesis put forth by Keitz et al. that the maximal value of the apparent rate constant for relaxation may serve as the kinetic definition of agonist efficacy was also tested and found to be invalid for the adenosine A2 receptor. We propose that this pair of activated receptors and tissue preparation is a good model to study quantitative aspects of functional antagonism by kinetic paradigms.

Kinetics of relaxation responses to vasorelaxants in isolated rabbit aorta.

Transient responses of isolated tissues to drugs are best studied by application of non-steady-state protocols in which the data collected are analyzed using kinetic models. The time dependence of the relaxation response of the adventitia- and endothelium-denuded rabbit aorta to four vasorelaxants (nitroglycerin, sodium nitroprusside, 5'-N-ethylcarboxamidoadenosine and isoproterenol) was analyzed by an exploratory kinetic model. A rapid relaxation (t1/2 = 1-3 min) was elicited by all vasorelaxants. An apparent desensitization or fade of the relaxation response to nitroglycerin or isoproterenol was visualized as the partial regaining of tissue tone (t1/2 = 2-3 min). The relaxation responses to sodium nitroprusside or 5'-N-ethylcarboxamidoadenosine were stable for at least 60 min and did not exhibit an apparent regaining of tension. Tissues rendered desensitized by either isoproterenol or nitroglycerin responded fully to sodium nitroprusside or 5'-N-ethylcarboxamidoadenosine. The rate constant for relaxation was vasorelaxant concentration-dependent and saturable for all vasorelaxants. For isoproterenol, nitroglycerin, and 5'-N-ethylcarboxamidoadenosine the rate constant for relaxation was inversely proportional to the contractile stimulus, as was the magnitude of relaxation for all vasorelaxants. Although the magnitude and rate constant of the fade was not concentration-dependent for isoproterenol, it was inversely proportional to the nitroglycerin concentration. The rate constant of the fade was proportional to the contractile stimulus for isoproterenol and nitroglycerin, and the magnitude of the fade was proportional to the contractile stimulus for nitroglycerin. We propose that kinetic studies of responses in isolated vasculature supersede studies performed under steady-state conditions, for they extend our knowledge of the manner by which the steady-state is achieved and allow for a quantitative analysis of the time-dependent changes which should assist in elucidating the biochemical basis of the observed physiological response.

Differential attenuation of the responses to adenosine and methoxamine in isolated rabbit aorta.

This article describes the functional antagonism between the responses to adenosine (through adenosine A2 receptors) and methoxamine (through alpha-1 adrenoceptors) in the adventitia- and endothelium-denuded isolated rabbit thoracic aorta. Rings were contracted with different concentrations of methoxamine and cumulative relaxation concentration-response curves (CRC) to adenosine were constructed. This protocol allowed the authors to rearrange the same data, which yielded contractile CRCs to methoxamine in the presence of adenosine. A 32-fold increase in the [methoxamine] markedly attenuated the maximal response to adenosine (80% decrease) and shifted the CRC to adenosine 10-fold to the right. By contrast, a 3000-fold increase in the [adenosine] shifted the CRC to methoxamine 3.25-fold to the right and attenuated the maximal response by a modest 18%. Analysis of these data by the operational model of agonism indicated that the efficacy parameter, tau, for adenosine or methoxamine was reduced by 99% or 71%, respectively, under these conditions. The agonist dissociation constant, KA, for adenosine (80 microM) or methoxamine (33 microM) by functional antagonism was also estimated. Use of an irreversible alpha-1 adrenoceptor antagonist allowed for the estimation of the KA for methoxamine by the receptor inactivation method using the operational model (40 microM), the Furchgott equation (48 microM) and the nested equations (42 microM) described by James et al. These results suggest that this tissue preparation is a good model to study functional antagonism quantitatively and that the functional antagonism between the responses mediated by these two receptors allows for the reliable estimation of the agonist dissociation constant for alpha-1 adrenoceptor agonists.