Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation.

Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle.

Electrical activity induced by nitric oxide in canine colonic circular muscle.

Nitric oxide generates slow electrical oscillations (SEOs) in cells near the myenteric edge of the circular muscle layer, which resemble slow waves generated by interstitial cells of Cajal (ICCs) at the submucosal edge of this muscle. The properties of SEOs were studied to determine whether these events are similar to slow waves. Rapid frequency membrane potential oscillations (MPOs; 16 +/- 1 cycles/min and 9.6 +/- 0.2 mV) were recorded from control muscles near the myenteric edge. Sodium nitroprusside (0.3 microM) reduced MPOs and initiated SEOs (1.3 +/- 0.3 cycles/min and 13.4 +/- 1.4 mV amplitude). SEOs were abolished by the guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (10 microM). MPOs were abolished by nifedipine (1 microM), whereas SEO frequency increased and the amount of depolarization decreased. BAY K 8644 (1 microM) prolonged SEOs and reduced their frequency. SEOs were abolished by Ni(2+) (0.5 mM), low Ca(2+) solution (0.1 mM Ca(2+)), cyclopiazonic acid (10 microM), and the mitochondrial uncouplers antimycin (10 microM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 microM). Oligomycin (10 microM) was without effect. These effects are similar to those described for colonic slow waves. Our results suggest that nitric oxide-induced SEOs are similar in mechanism to slow waves, an activity not previously thought to be generated by myenteric pacemakers.

Development of an ELISA screening test for nitroimidazoles in egg and chicken muscle

An antibody was generated that can bind metronidazole (MNZ), a nitroimidazole drug used in veterinary medicine to treat poultry for coccidiosis and histomoniasis. A direct competitive enzyme-linked immunosorbent assay (cELISA) is described. It was used to characterise binding of this antibody to a number of nitroimidazole drugs. It displayed cross-reactivity with dimetridazole (DMZ), ronidazole (RNZ), hydroxydimetridazole (DMZOH), and ipronidazole (IPZ).