A highly sensitive and versatile virus titration assay in the 96-well microplate format.

This report describes a fast, reproducible, inexpensive and convenient assay system for virus titration in the 96-well format. The micromethod substantially increases assay throughput and improves the data reproducibility. A highly simplified variant of virus quantification is based on immunohistochemical detection of virus amplification foci obtained without use of agarose or semisolid overlays. It can be incorporated into several types of routine virological assays successfully replacing the laborious and time-consuming conventional methods based on plaque formation under semisolid overlays. The method does not depend on the development of CPE and can be accommodated to assay viruses with substantial differences in growth properties. The use of enhanced immunohistochemical detection enabled a five- to six-fold reduction of the total assay time. The micromethod was specifically developed to take advantage of multichannel pipettor use to simplify handling of a large number of samples. The method performs well with an inexpensive low-power binocular, thus offering a routine assay system usable outside of specialized laboratory setting, such as for testing of clinical or field samples. When used in focus reduction-neutralization tests (FRNT), the method accommodates very small volumes of immune serum, which is often a decisive factor in experiments involving small rodent models.

Reduced effectiveness of HMR 1098 in blocking cardiac sarcolemmal K(ATP) channels during metabolic stress.

ATP-sensitive K(+) (K(ATP)) channels are involved in ischemic cardioprotection induced by preconditioning (IPC), though the relative role of sarcolemmal (sK(ATP)) and mitochondrial (mitoK(ATP)) channels remains controversial. The sK(ATP)-selective sulphonylthiourea HMR 1098 has often been reported to be without effect on ischemic cardioprotection, suggesting minimal involvement of sK(ATP). Since some sulphonylureas show reduced potency under conditions of metabolic stress, we used patch clamp to assess the ability of HMR 1098 to block sK(ATP) currents of adult rat ventricular myocytes activated by metabolic inhibition (MI, NaCN+iodoacetate). In contrast to the prototype sulphonylurea glibenclamide, HMR 1098 (10 muM) was without effect on sK(ATP) currents, and also did not inhibit MI-induced action potential shortening. However, HMR 1098 blocked sK(ATP) current induced by the K(ATP) opener pinacidil (IC(50)=0.36+/-0.02 muM), and reversed pinacidil-induced action potential shortening. In inside-out patches, block by HMR 1098 was relieved by increasing MgADP concentrations (1-100 muM). HMR 1098 inhibited pinacidil-activated recombinant Kir6.2/SUR2A channels with a similar IC(50) (0.30+/-0.04 muM), but was less effective when channels were activated by low intracellular ATP. HMR 1098 displaced binding of the pinacidil analogue [(3)H]P1075 to native cardiac membranes with a biphasic inhibition curve. Our results show that HMR 1098 becomes a much less effective inhibitor of sK(ATP) during metabolic stress, and suggest that the lack of effect of HMR 1098 on ischemic cardioprotection reported in some studies may represent loss of block by the drug under these conditions rather than a lack of involvement of sK(ATP) channels.

Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes.

The subcellular distribution of ATP-sensitive potassium (K(ATP)) channel subunits in rat-isolated ventricular myocytes was investigated using a panel of subunit-specific antisera. Kir6.1 subunits were associated predominantly with myofibril structures and were co-localized with the mitochondrial marker MitoFluor red (correlation coefficient (cc) = 0.63 +/- 0.05). Anti-Kir6.1 antibodies specifically recognized a polypeptide of 48 kDa in mitochondrial membrane fractions consistent with the presence of Kir6.1 subunits in this organelle. Both Kir6.2 and SUR2A subunits were distributed universally over the sarcolemma. Lower-intensity antibody-associated immunofluorescence was detected intracellularly, which was correlated with the distribution of MitoFluor red in both cases (cc, Kir6.2, 0.56 +/- 0.05; SUR2A, 0.61 +/- 0.06). A polypeptide of 40 kDa was recognized by anti-Kir6.2-subunit antibodies in western blots of both microsomal and mitochondrial membrane fractions consistent with the presence of this subunit in the sarcolemma and mitochondria. Similarly, SUR2A and SUR2B subunits were detected in western blots of microsomal membrane proteins consistent with a sarcolemmal localization for these polypeptides. SUR2B subunits were shown in confocal microscopy to co-localize strongly with t-tubules (cc, 0.81 +/- 0.05). Together, the results indicate that Kir6.2 and SUR2A subunits predominate in the sarcolemma of ventricular myocytes consistent with a Kir6.2/SUR2A-subunit combination in the sarcolemmal K(ATP)channel. Kir6.1, Kir6.2 and SUR2A subunits were demonstrated in mitochondria. Combinations of these subunits would not explain the reported pharmacology of the mitochondrial K(ATP) channel (Mol Pharmacol 59 (2001) 225) suggesting the possibility of further unidentified components of this channel.

K(ATP) channels mediate the beta(2)-adrenoceptor agonist-induced relaxation of rat detrusor muscle.

We propose that ATP-sensitive K(+) (K(ATP)) channels are normally inactive but involved in beta(2)-adrenoceptor stimulated relaxation of the rat bladder. Spontaneous detrusor muscle contractions were unaffected by glibenclamide (K(ATP) channel blocker) but were reduced when pinacidil (K(ATP) channel opener) concentrations exceeded 10(-5) M. Inhibition by beta(2)-adrenoceptor agonist clenbuterol [10(-6) M] of 1 Hz electrical field stimulated contractions was abolished by glibenclamide [10(-6) M]. Glibenclamide [10(-6) M] decreased forskolin-induced relaxation [10(-9)-10(-4) M] in bladder muscle stimulated with 1 Hz electrical field. In the presence glibenclamide (10(-6) M) or myristoylated protein kinase A inhibitor (2)x[10(-6) M], clenbuterol [10(-9)-10(-5) M] failed to inhibit bladder contraction in response to 1 Hz electrical field stimulation. Therefore, K(ATP) channel opening and the subsequent hyperpolarization of cell membranes in response to beta(2)-adrenoceptor activation is mediated by raised cyclic-AMP levels and activation of protein kinase A. This counteracts ATP-stimulated depolarization in bladder muscle, thereby reducing cell contraction.

Inhibition of the contractile response of the rat detrusor muscle by the beta(2)-adrenoceptor agonist clenbuterol.

The action of clenbuterol, beta(2)-adrenoceptor agonist, on the contractile response of isolated rat detrusor muscle strips was investigated in vitro. Clenbuterol (10(-5) M) inhibited the detrusor muscle frequency response (1-40 Hz, p<0.02) with a more pronounced effect at 1 Hz than 40 Hz. Clenbuterol (10(-6) M) significantly inhibited the contractile response to exogenous ATP (10(-4) to 10(-2) M, p<0.05) but not to carbachol (10(-9) to 10(-4) M). The presence of 10(-5) M ICI 118, 551, beta(2)-adrenoceptor antagonist, shifted significantly the clenbuterol dose-response to 1 Hz electrical field stimulation (EC(50) 3.4x10(-6) M (+/-2.2x10(-6) M) for clenbuterol alone, to 4.1x10(-4) M (+/-8.8 x10(-5) M), P<0.05). In conclusion, clenbuterol inhibits electrical field and ATP-stimulated contractions of detrusor muscle. Reversal of the clenbuterol inhibition of detrusor muscle contraction by ICI 118, 551 shows that clenbuterol is probably acting through postsynaptic beta(2)-adrenoceptors, which modulate the response to ATP released from purinergic nerves.