Mechanoregulation of BK channel activity in the mammalian cortical collecting duct: role of protein kinases A and C

Flow-stimulated net K secretion ( JK) in the cortical collecting duct (CCD) is mediated by an iberiotoxin (IBX)-sensitive BK channel, and requires an increase in intracellular Ca2+ concentration ([Ca2+]i). The α-subunit of the reconstituted BK channel is phosphorylated by PKA and PKC. To test whether the BK channel in the native CCD is regulated by these kinases, JK and net Na absorption ( JNa) were measured at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in the presence of mPKI, an inhibitor of PKA; calphostin C, which inhibits diacylglycerol binding proteins, including PKC; or bisindolylmaleimide (BIM) and Gö6976, inhibitors of classic and novel PKC isoforms, added to luminal (L) and/or basolateral (B) solutions. L but not B mPKI increased JK in CCDs perfused at a slow flow rate; a subsequent increase in flow rate augmented JK modestly. B mPKI alone or with L inhibitor abolished flow stimulation of JK. Similarly, L calphostin C increased JK in CCDs perfused at slow flow rates, as did calphostin C in both L and B solutions. The observation that IBX inhibited the L mPKI- and calphostin C-mediated increases in JK at slow flow rates implicated the BK channel in this K flux, a notion suggested by patch-clamp analysis of principal cells. The kinase inhibited by calphostin C was not PKC as L and/or B BIM and Gö6976 failed to enhance JK at the slow flow rate. However, addition of these PKC inhibitors to the B solution alone or with L inhibitor blocked flow stimulation of JK. Interpretation of these results in light of the effects of these inhibitors on the flow-induced elevation of [Ca2+]i suggests that the principal cell apical BK channel is tonically inhibited by PKA and that flow stimulation of JK in the CCD is PKA and PKC dependent. The specific targets of the kinases remain to be identified.

Influence of liner design on mouthpiece chamber vacuum during milking

SummaryThree types of liner (wide bore tapered, wide bore and narrow bore) were compared in a change-over design experiment comprising three 3-d periods and using six lactating Holstein Friesian cows with a wide range in milk flow rate and teat dimensions. Vacuum levels measured within the liner showed a characteristic pattern of three distinct phases: 1, a mouthpiece chamber (MPC) vacuum that fluctuated with vacuum beneath the teat; 2, a stable MPC vacuum that coincided with the period of peak milk flow and 3, a MPC vacuum that fluctuated with pressure changes in the pulsation chamber and coincided with the period of no or reduced flow. Of the three liners examined, the Gascoigne 1030A (wide bore tapered) induced the highest MPC vacuum (29·4 ± 2·23 kPa, mean ± s.e.m), significantly higher (P < 0·01) than the Alfa-Laval 960000–01 wide bore (23·5 ± 2·32), regardless of teat size and milk flow rate. The Alfa-Laval 7275737–01 (narrow bore) gave a significantly lower value (16·5 ± 1·96) than either of the others (P < 0·001). It was not possible to link absolutely raised MPC vacuum to cow discomfort and teat trauma but circumstantial evidence encourages this view. Narrow teats in combination with slow flow rate appeared to exacerbate MPC vacuum and, because milking time for such animals was protracted, teats were exposed for longer periods.