Chondrocytes possess the capacity to transduce load-induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch-clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell-attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20–200 mmHg) activated ion channel currents in six of seven patches. The mean activated current was 45.9 ± 1.1 pA (n = 4) at a membrane potential of 33 mV (cell surface area approximately 240 µm2). The mean slope conductance of the principal single channels resolved within the total stretch-activated current was 118 ± 19 pS (n = 6), and reversed near the theoretical potassium equilibrium potential, EK+, suggesting it was a high-conductance potassium channel. Activation of these high-conductance potassium channels was inhibited by extracellular TEA (Kd approx. 900 µM) and iberiotoxin (Kd approx. 40 nM). This suggests that the current was largely carried by BK-like potassium (MaxiK) channels. To further characterize these BK-like channels, we used inside-out patches of chondrocyte membrane: we found these channels to be activated by elevation in bath calcium concentration. Immunohistochemical staining of equine cartilage samples with polyclonal antibodies to the α1- and β1-subunits of the BK channel revealed positive immunoreactivity for both subunits in superficial zone chondrocytes. These experiments support the hypothesis that functional BK channels are present in chondrocytes and may be involved in mechanotransduction and chemotransduction.

No abstract

The aquaporins (AQPs) are a family of small integral membrane proteins 28-35kDa in size, sub classified into 2 groups; those selectively permeated by water (the aquaporins; AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8) or by water and small organic osmolytes such as glycerol and urea (the aquaglyceroporins; AQP3, AQP7, AQP9 and AQP10). AQPs are expressed in a variety of epithelial tissues where they are responsible for rapid water movement driven by osmotic gradients. The mammary gland produces and secretes milk which consists of water, lipids, electrolytes, vitamins, sugars and specific milk proteins. However, little is known about AQP expression, distribution and function in mammary tissue or about the process of water transport across the mammary epithelium. The only information available relates to the identification of AQP1 and AQP3 in capillary endothelia and epithelial cells of mouse mammary glands respectively (Matsuzaki et al., 2005). Since water delivery to the mammary gland is critical for milk production, which is of major economic importance to the dairy industry, it is necessary to understand the underlying physiology involved. The aim of this study was to determine the expression and distribution of AQPs within the bovine mammary gland.

In this study we used custom‐designed rabbit polyclonal antisera to examine the expression and cellular localization of aquaporins along the secretory pathway of actively lactating bovine mammary glands by immunohistochemistry. Mammary tissues examined included secretory ducts and acini, gland cisterns, teats, connective and adipose tissues. AQP1 was found in capillary endothelia throughout the lactational mammary gland in addition to myoepithelial cells underlying teat duct epithelia. Expression of AQP2 and AQP6 was not detected and AQP9 was only found in leukocytes. AQP3 and AQP7 were found in smooth muscle bundles in the teat, secretory epithelial cells and duct epithelial cells. AQP4 was observed in selected epithelial cells in the teat, cistern and secretory acini. AQP5 expression was confined to acini and glandular structures. These immunohistochemical findings support a functional role for aquaporins in the transport of water and small solutes across endothelial and epithelial barriers in the mammary gland and in the production and secretion of milk.